by Kwong Joseph. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 142-164). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Prostatic Secretory Proteins --- p.1 / Chapter 1.2 --- Rat Prostatic Secretory Proteins --- p.1 / Chapter 1.2.1 --- Prostatic Secretory Proteins in Ventral Prostate --- p.2 / Chapter 1.2.1.1 --- Prostatic Binding Protein (PBP) --- p.2 / Chapter 1.2.1.2 --- Androgen-Suppressed Proteins of Rat Ventral Prostate --- p.6 / Chapter 1.2.1.3 --- The 20-kDa Protein --- p.8 / Chapter 1.2.1.4 --- Spermine-Binding Proteins --- p.9 / Chapter 1.2.1.5 --- Prostatic Acid Phosphatase (PAP) --- p.10 / Chapter 1.2.2 --- Prostatic Secretory Proteins in Dorsal Prostate --- p.12 / Chapter 1.2.2.1 --- Dorsal Proteins I and II (DP I and DPII) --- p.12 / Chapter 1.2.2.2 --- Seminal Vesicle Secretion II (SVSII) --- p.14 / Chapter 1.2.2.3 --- Probasin --- p.16 / Chapter 1.2.3 --- Prostatic Secretory Proteins in Lateral Prostate --- p.18 / Chapter 1.3 --- Human Prostatic Secretion --- p.18 / Chapter 1.4 --- Human Prostatic Secretory Proteins --- p.18 / Chapter 1.4.1 --- Prostatic Acid Phosphatase (PAP) --- p.19 / Chapter 1.4.2 --- Prostate Specific Antigen (PSA) --- p.22 / Chapter 1.4.2.1 --- Molecular Biology of PSA --- p.22 / Chapter 1.4.2.2 --- Synthesis of PSA --- p.23 / Chapter 1.4.2.3 --- Kallikrein Gene Family --- p.23 / Chapter 1.4.2.4 --- Physiological Function of PSA --- p.24 / Chapter 1.4.2.5 --- PSA as an Immunohistochemical Marker --- p.25 / Chapter 1.4.2.6 --- PSA is not a Prostate-Specific Molecule --- p.26 / Chapter 1.4.3 --- Prostatic Secretory Protein of 94 Amino Acids (PSP94) --- p.27 / Chapter 1.4.3.1 --- Nucleotide Sequence of the PSP94 cDNA --- p.28 / Chapter 1.4.3.2 --- Amino Acid sequence of PSP94 --- p.28 / Chapter 1.4.3.3 --- Biological Properties of PSP94 --- p.29 / Chapter 1.4.3.4 --- Physiological Roles of PSP94 --- p.31 / Chapter 1.4.3.5 --- PSP94 and Its mRNA in Other Non-Prostatic Tissue --- p.31 / Chapter 1.4.3.6 --- PSP94 as a Tumor Marker of Prostate Cancer --- p.32 / Chapter 1.4.3.7 --- Homologous Proteins of PSP94 --- p.34 / Chapter 1.5 --- Aim of Study --- p.35 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and Supply of Noble Rat --- p.37 / Chapter 2.2 --- Chemicals --- p.37 / Chapter 2.3 --- Bilateral Ochidectomy of Animals --- p.37 / Chapter 2.4 --- Androgen Replacement --- p.38 / Chapter 2.5 --- Hormonal and Drug Treatments on Castrated Animals --- p.38 / Chapter 2.6 --- Induction of Prostatic Intraepithelial Neoplasia in Noble Rat Prostate Gland by Long-Term Treatment with Steroids --- p.39 / Chapter 2.6.1 --- Preparation of Steroid Hormone-Filled Silastic® Tubings --- p.39 / Chapter 2.6.2 --- Surgical Implantation of Silastic® Tubings --- p.39 / Chapter 2.6.3 --- Protocols of Hormonal Treatments --- p.40 / Chapter 2.7 --- Androgen-Dependent Rat Dunning Prostatic Adenocarcinoma --- p.40 / Chapter 2.8 --- Androgen-Independent Prostatic Carcinoma Line (AIT) of Noble Rat --- p.41 / Chapter 2.9 --- Plasmids --- p.41 / Chapter 2.10 --- Restriction Enzyme Digestions of pLvB10 and cM-403 --- p.42 / Chapter 2.11 --- Amplification of Rat SVSII cDNA Fragment by RT-PCR and Subcloning --- p.42 / Chapter 2.12 --- Purification of DNA Fragment from Agarose Gel --- p.43 / Chapter 2.13 --- Subcloning of DNA into Vector --- p.44 / Chapter 2.14 --- Tissue Preparation for In-situ Hybridization --- p.47 / Chapter 2.15 --- Synthesis of Digoxigenin (DIG)-Labeled RNA Probe --- p.47 / Chapter 2.16 --- In-situ Hybridization --- p.48 / Chapter 2.17 --- Total RNA Extraction --- p.50 / Chapter 2.18 --- Northern Blotting Analysis --- p.51 / Chapter 2.19 --- Primers and Cycling Conditions --- p.53 / Chapter 2.20 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.54 / Chapter 2.21 --- Southern Blotting Analysis --- p.56 / Chapter 2.21.1 --- Southern Blotting --- p.56 / Chapter 2.21.2 --- Preparation of DIG-dUTP Labeled Rat PSP94 cDNA Probe --- p.56 / Chapter 2.21.3 --- Hybridization --- p.57 / Chapter 2.22 --- Restriction Mapping --- p.58 / Chapter 2.23 --- Semi-Quantitative RT-PCR --- p.59 / Chapter 2.24 --- Statistical Analysis --- p.59 / Chapter 2.25 --- "Protein Extraction, SDS-PAGE and Western Blotting Analysis" --- p.60 / Chapter 2.26 --- Immunohistochemistry --- p.63 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Subcloning of DNAs into Vector --- p.65 / Chapter 3.1.1 --- Subcloning of 18s Ribosomal RNA cDNA Fragment --- p.65 / Chapter 3.1.2 --- Subcloning of Probasin cDNA Fragment --- p.66 / Chapter 3.1.3 --- Subcloning of SVSII cDNA Fragment --- p.66 / Chapter 3.1.4 --- Restriction Enzyme Mapping for PCR Product of SVSII --- p.67 / Chapter 3.2 --- Detection of mRNA and Protein Expression of PSP94 in Normal Rat Prostates --- p.68 / Chapter 3.2.1 --- In-situ Hybridization --- p.68 / Chapter 3.2.2 --- Northern Blotting --- p.68 / Chapter 3.2.3 --- RT-PCR Amplification --- p.69 / Chapter 3.2.4 --- Immunohistochemistry --- p.69 / Chapter 3.2.5 --- Western Blotting --- p.70 / Chapter 3.3 --- Detection of mRNA Expression of Probasin and SVSII in Normal Rat Prostates --- p.71 / Chapter 3.3.1 --- In-situ Hybridization --- p.71 / Chapter 3.3.2 --- RT-PCR Amplification --- p.71 / Chapter 3.4 --- "Androgen Regulation of PSP94,Probasin and SVSII mRNA Expression" --- p.72 / Chapter 3.4.1 --- In-situ Hybridization --- p.72 / Chapter 3.4.2 --- "Relative Expression Levels of PSP94, Probasin and SVSII mRNA in Normal, Castrated and Androgen Replaced Rat Lateral Prostates as Measured by a Semiquantitative RT-PCR Method" --- p.73 / Chapter 3.4.2.1 --- Determination of Exponential Range of PCR --- p.73 / Chapter 3.4.2.2 --- Semi-Quantitative RT-PCR --- p.74 / Chapter 3.4.3 --- Western Blot Analysis --- p.75 / Chapter 3.5 --- "Effect of Steroid Hormones and Zinc on the PSP94, Probasin and SVSII Expressions in Castrated Rat Prostates" --- p.76 / Chapter 3.5.1 --- Semi-Quantitative RT-PCR --- p.76 / Chapter 3.5.2 --- Western Blot Analysis --- p.77 / Chapter 3.6 --- "Detection of PSP94, Probasin and SVSII mRNA Expression in Dysplastic and Neoplastic Rat Prostates" --- p.78 / Chapter 3.6.1 --- "Detection of PSP94, Probasin and SVSII mRNA Expression in T+E2-Induced Prostatic Intraepithelial Neoplasia (PIN) of the Lateral Prostate of Noble Rats by In-situ Hybridization" --- p.78 / Chapter 3.6.2 --- "Detection of PSP94,Probasin and SVSII mRNA Expression in Dunning Tumor and AIT Prostatic Tumor" --- p.79 / Chapter 3.6.2.1 --- In-situ Hybridization --- p.79 / Chapter 3.6.2.2 --- RT-PCR Amplification --- p.79 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Specific Expression of PSP94 in the Lateral Lobe of Rat Prostate --- p.114 / Chapter 4.2 --- Androgen Regulation of PSP94 --- p.118 / Chapter 4.2.1 --- Molecular Mechanism of Androgen Action --- p.118 / Chapter 4.2.2 --- Androgen Regulation of PSP94 in Rat Lateral Prostate --- p.121 / Chapter 4.3 --- "Effect of Steroid Hormones and Zinc on the PSP94, Probasin and SVSII Expressions in Castrated Rat Lateral Prostate" --- p.126 / Chapter 4.4 --- "Detection of PSP94, Probasin, SVSII mRNA Expression in Dysplastic and Neoplastic Rat Prostates" --- p.133 / Chapter 4.5 --- Gene Therapy --- p.139 / Chapter Chapter 5 --- Conclusions --- p.141 / References --- p.142 / Appendixes --- p.165

Chan Chi-keung. / Thesis (M.Sc.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 93-107). / Abstracts in English and Chinese. / List of Tables --- p.ix / List of Figures --- p.x / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Oncogenic anatomy of the prostate gland --- p.1 / Chapter 1.3 --- Characteristics of prostate cancer --- p.7 / Chapter 1.4 --- Incidences of prostate cancer --- p.8 / Chapter 1.5 --- Risk factors for prostate cancer --- p.14 / Chapter 1.5.1 --- Endogenous risk factors --- p.14 / Chapter (A) --- Age --- p.14 / Chapter (B) --- Race --- p.16 / Chapter (C) --- Family history --- p.21 / Chapter (D) --- Hormonal factors --- p.24 / Chapter (I) --- Androgen --- p.24 / Chapter (II) --- Vitamin D --- p.32 / Chapter 1.5.2 --- Exogenous risk factors --- p.41 / Chapter (A) --- Dietary factors --- p.41 / Chapter (B) --- Body Mass Index & physical condition --- p.44 / Chapter (C) --- Occupation --- p.46 / Chapter (D) --- Vasectomy --- p.47 / Chapter (E) --- Others --- p.48 / Chapter 2. --- Introduction to the project --- p.49 / Chapter 3. --- Objectives --- p.50 / Chapter 4. --- Materials and Methods --- p.51 / Chapter 4.1 --- Prostate cancer cases --- p.51 / Chapter 4.2 --- Controls --- p.52 / Chapter (A) --- Benign prostatic hyperplasia --- p.52 / Chapter (B) --- Population control --- p.52 / Chapter 4.3 --- DNA extraction --- p.53 / Chapter 4.4 --- Amplification of target DNA --- p.54 / Chapter 4.5 --- Allele typing --- p.55 / Chapter 4.6 --- Statistical analysis --- p.55 / Chapter 5. --- Results --- p.60 / Chapter 5.1 --- Optimization of DNA extraction --- p.60 / Chapter 5.2 --- Optimization of PCR condition --- p.61 / Chapter 5.3 --- Allele typing --- p.65 / Chapter 5.4 --- Characteristics of subjects samples --- p.68 / Chapter 5.4.1 --- Age of subjects and tumor grading --- p.68 / Chapter 5.4.2 --- Genotype typing --- p.69 / Chapter (A) --- Bsm genotype --- p.69 / Chapter (B) --- Fok genotype --- p.69 / Chapter 6. --- Discussions --- p.73 / Chapter 6.1 --- Technical issues --- p.73 / Chapter (A) --- DNA extraction --- p.73 / Chapter (B) --- Primer design --- p.76 / Chapter (C) --- Determination of the optimal PCR condition --- p.77 / Chapter (D) --- Restriction enzyme digestion --- p.82 / Chapter 6.2 --- Age distribution of prostate cancer patients --- p.83 / Chapter 6.3 --- Genotype frequency --- p.84 / Chapter 6.4 --- Histopathological samples of case and control --- p.87 / Chapter 6.5 --- Vitamin D receptor genotypes and prostate cancer --- p.89 / Chapter 7. --- Conclusions --- p.92 / Chapter 8. --- References --- p.93

Background and aims of the study. Prostate cancer (PCa) is one of the most common hormone-dependent cancers in men in Western and also Asian countries. The standard treatment options for localized PCa include surgery and androgen-deprivation therapy (ADT). However, most patients upon ADT therapy invariably relapse and progress to a more aggressive and metastatic stage termed as castration-resistant PCa (CRPC). Accumulating studies indicate that androgen receptor (AR) transcriptional activity is dysregulated during the advanced progression of CRPC. One important mechanism responsible for the growth of CRPC includes increased intra-tumoral androgen synthesis in PCa. Recently, a novel androgen-responsive fusion gene TMPRSS2:ERG formed by fusion between the transmembrane protein TMPRSS2 and transcription factor ERG, has been identified in approximately 50% PCa samples, which results in the aberrant expression of ERG function as oncogenic factor in PCa. Currently, TMPRSS2:ERG is regarded as a significant potential diagnostic and prognostic biomarker for PCa. Estrogen-related receptor alpha-ERRα, the first identified ligand-independent orphan nuclear receptor, is characterized to be up-regulated in advanced cancers, suggesting that ERRα might play important regulatory roles in the malignant progression of PCa. Previous studies showed that ERRα can functionally cross-talk with AR signaling via co-targeting to AR targets and regulate the expression of some steroidogenic enzymes in breast cancer. Based on this background, it is hypothesized that ERRα could functionally regulate the TMPRSS2:ERG fusion gene and play a regulatory role in the development and progression of CRPC through activation of the intracellular androgen synthesis pathway. / Results. 1) The results obtained in this study showed that suppression of ERRα by its specific inverse agonist XCT790 or shRNA-knockdown could induce down-regulation of TMPRSS2:ERG and also its target genes in AR-positive VCaP PCa cells. 2) Ectopic expression of ERRα and/or its coactivator PGC-1α could increase the expression of TMPRSS2:ERG in AR-negative NCI-H660 PCa cells. 3) Two ERRα-DNA binding elements were identified by ChIP assay and sequence analysis in the promoter of TMPRSS2:ERG and both of these two elements could be transactivated by ERRα and PGC-1α. 4) Ectopic expression of TMPRSS2:ERG under the regulation of ERRα enhanced the prostatic cell invasion capacity as shown in the TMPRSS2:ERG infectants of BPH-1 and PC-3 prostatic cells. 5) ERG expressed by the TMPRSS2:ERG fusion could directly transactivate the ERRα gene in prostatic cells. 6) A positive correlation on the expressions between TMPRSS2:ERG and ERRα was demonstrated in a xenograft model of CRPC (VCaP-CRPC). 7) The expression of TMPRSS2:ERG and ERRα showed significant up-regulation and the transactivation activity of ERRα was also enhanced in castration-resistant VCaP-CRPC cells. 8) Ectopic expression of ERRα could promote resistant growth capacity to androgen-deprivation condition in LNCaP PCa cells, whereas shRNA-mediated silence of ERRα could weaken this resistant capacity. Furthermore, ectopic expression of ERRα in LNCaP-ERRα infectants could promote their in vivo growth resistance to castration in SCID mice. 9) Expression of several androgenic enzyme genes, including CYP11A1, CYP17A1 and ARK1C3, were detected to be up-regulated in castration-resistant VCaP-CRPC cells. Moreover, ectopic expression of ERRα could induce the increased expression of these enzyme genes in LNCaP-ERRα infectants, whereas knockdown of ERRα by shRNA could decrease their expression. 10) ERRα could directly transactivate the gene promoters of CYP11A1, CYP17A1 and ARK1C3 which contain ERRE elements prediction by sequence analysis. These results suggested that ERRα could play a role in de novo or intra prostatic androgen synthesis in the PCa cells. / Conclusions. The results obtained in this study suggested that ERRα and TMPRSS2:ERG could form a positive reciprocal loop in PCa cells, and ERRα could also promote the resistant growth capacity of PCa cells resistant to the androgen-deprivation condition in vitro and also castration-resistant growth in vivo via a mechanism of up-regulation of androgenic enzyme genes. The results also suggested that ERRα might play a significant regulatory role in the development and progression of PCa, particularly the advanced CRPC, and also ERRα could be a potential therapeutic target for the treatment of PCa, particularly the advanced PCa-CRPC. / 研究背景與研究目的：前列腺癌作為激素依賴的一種癌症，經常出現在西方和亞洲國家的男性人群中。對於局限性前列腺癌多採用外科手術和去勢的治療。但是大多數病人經過去勢治療后會再次復發並且形成更加惡心幾轉移的前列腺癌，稱之為去勢難治性前列腺癌（CRPC）。越來越多的研究表明在去勢難治性前列腺癌發病過程中，雄激素受體轉錄活性異性增強。其中一個重要機理解釋為前列腺癌細胞自身合成的雄激素增多。進來，在大約50%的前列腺癌病人中新檢測到一個受雄激素受（AR）體調控的融合基因TMPRSS2:ERG，它是由稱為TMPRSS2的一個跨膜蛋白和一個稱為ERG的轉錄因子融合而成，它的出現導致了在前列腺癌中異常的稱為致癌因子的ERG蛋白的高表達。目前，TMPRSS2:ERG已經被作為一個重要的潛在的診斷和預測的標誌物應用在前列腺癌中。作為第一個鑒定的配體不依賴的孤兒受體-ERRα，被證明在晚期的癌症中有很高的表達，預示著ERRα可能在惡性的癌症中起到一個非常重要的調控作用。之前的研究表明通過共同調控AR的下游基因，ERRα同AR信號通路之間有功能性的交叉調控；除此之外，在乳腺癌中，ERRα還可以調控一些類固醇類化合物的合成相關的一些酶的合成。依據上述，我們推定ERRα可能功能性地調控TMPRSS2:ERG融合基因的表達並且通過調控細胞內的雄激素的合成進而在去勢難治性前列腺癌的發生和發展中起到一個非常重要的作用。 / 結果：本論文研究結果總結如下：1）在有AR表達的前列腺癌細胞-VCaP細胞中，通過ERRα特異性的抑制劑XCT790處理或者shRNA介入的干擾ERRα的mRNA的方法來抑制ERRα，下調了TMPRSS2:ERG和它的一些下游調控基因的表達。2）在沒有AR表達的前列腺癌細胞-NCI-H660細胞中，上調ERRα或者它的特異性的共激活因子PGC-1α表達可以提升TMPRSS2:ERG的表達。3）通過ChIP實驗，在TMPRSS2:ERG的啟動子上面，兩個ERRα的DNA結合位點被鑒定出來。並且這兩個位點可以被ERRα和PGC-1α轉錄激活。4）在兩個前列腺細胞BPH-1和PC-3細胞中，在ERRα的調控下高表達TMPRSS2:ERG融合基因可以增強細胞的侵襲能力。5）融合基因TMPRSS2:ERG導致的ERG蛋白的表達可以直接轉錄激活ERRα的表達。6）我們通過VCaP細胞的異種移植建立VCaP-CRPC的體內模型來模擬CRPC過程，在整個過程中，我們發現TMPRSS2:ERG和ERRα有一致性的表達相關性。除此之外，我們根據上述動物模型通建立了VCaP-CRPC細胞系，並且發現在VCaP-CRPC細胞細胞中，TMPRSS2:ERG和ERRα都有被上調並且ERRα的轉錄活性同樣也提升。7）在LNCaP細胞中高表達ERRα可以提升細胞在去除雄激素的環境中生長的能力。但是當在LNCaP細胞中用shRNA干擾掉ERRα可以明顯減弱這種生長的能力。用LNCaP-ERRα穩轉ERRα的細胞異種移植建立SCID老鼠體內腫瘤模型，我們發現和LNCaP-pBABE對照組相比，LNCaP-ERRα細胞生長的更快更大。並且在對老鼠進行睪丸切除術后，LNCaP-ERRα組細胞更快適應這種環境并繼續生長，相比之下，LNCaP-pBABE對照組則持續萎縮減小。8）在上述的VCaP-CRPC細胞中，我們發現一些和雄激素合成相關的關鍵的酶包括CYP11A1，CYP17A1和ARK1C3的表達量有顯著地提升。而且在LNCaP-ERRα細胞中同樣檢測到這些酶的表達量的提升。然而當在LNCaP細胞中用shRNA干擾掉ERRα可以明顯減降低上述酶的表達。9）我們在CYP11A1，CYP17A1和ARK1C3基因的啟動子區域發現有ERRα結合位點，並且發現這些位點可以被ERRα轉錄激活。 / 結論：本論文的研究結果提示在前列腺癌細胞中，ERRα和TMPRSS2:ERG可以形成一個相互正向調控的循環。除此之外，上調ERRα可以促進細胞在去除雄激素的環境中生長的能力，並且在動物體內可以提升細胞在睪丸去除的環境中的適應和生長能力。這種體內和體外的能力的提升是通過一種潛在的上調前列腺癌細胞的雄激素合成相關的關鍵的酶的表達，進而提升雄激素的含量而得以實現的。上述的結果預示著ERRα可能在前列腺癌發生機發展的過程中起到非常重要的調控作用，尤其在晚期的CRPC中。同時，ERRα也可能作為一個潛在的重要的前列腺癌尤其是晚期的CRPC的治療靶點，尤其是一些潛在ERRα的特異性抑制劑，比如XCT790，可能作為將來用以作為治療前列腺癌的特異性靶點藥物。 / Xu, Zhenyu. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 126-143). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / Xu, Zhenyu. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

by Lau Hoi Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 83-96). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Endocrine control of normal and abnormal growth of prostate --- p.1 / Chapter 1.1.1 --- Androgen regulation of prostate gland --- p.1 / Chapter 1.1.2 --- Estrogen regulation of prostate gland --- p.4 / Chapter 1.2 --- Gonadotropin-releasing hormone plays a central role in reproduction --- p.6 / Chapter 1.2.1 --- GnRH gene --- p.7 / Chapter 1.2.2 --- GnRH receptor --- p.9 / Chapter 1.3 --- Therapeutic strategies using GnRH analogs to treat prostate cancer --- p.12 / Chapter 1.4 --- Expression of GnRH or its receptor in reproductive tissues --- p.12 / Chapter 1.4.1 --- Expression of GnRH in reproductive --- p.13 / Chapter 1.4.2 --- Expression of GnRH and its receptor in pituitary and reproductive tissues --- p.13 / Chapter 1.5 --- Animal models for the study of prostate cancer --- p.15 / Chapter 1.5.1 --- Nobel rat inducible model --- p.15 / Chapter 1.5.2 --- Androgen dependent rat Dunning prostatic tumor --- p.16 / Chapter 1.5.3 --- Androgen-independent prostatic carcinoma line of Noble rat --- p.18 / Chapter 1.6 --- Aim of study --- p.18 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and supply of Nobel rat --- p.20 / Chapter 2.2 --- Induction of dysplasia in Nobel rat prostate gland by long-term treatment with steroids --- p.20 / Chapter 2.2.1 --- Chemicals --- p.20 / Chapter 2.2.2 --- Preparation of steroid hormone-filled Silastic tubings --- p.20 / Chapter 2.2.3 --- Surgical implantation of Silastic® tubings --- p.21 / Chapter 2.2.4 --- Protocols of hormonal treatments --- p.21 / Chapter 2.3 --- Androgen- dependent Dunning rat prostatic adenocarcinoma --- p.22 / Chapter 2.4 --- Androgen- independent prostatic carcinoma line (ALT) of Noble rat --- p.22 / Chapter 2.5 --- Detection of mRNA expression of gonadotropin- releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.23 / Chapter 2.5.1 --- Preparation of tissue for total RNA extraction --- p.23 / Chapter 2.5.2 --- Total RNA extraction --- p.24 / Chapter 2.5.3 --- Reverse-transcription Polymerase Chain Reaction (RT-PCR) --- p.25 / Chapter 2.5.4 --- Purification of DNA fragments from agarose gels --- p.27 / Chapter 2.5.5 --- Subcloning of DNA into vector --- p.27 / Chapter 2.5.6 --- Nucleotide sequencing --- p.30 / Chapter 2.5.7 --- Southern blot analysis --- p.32 / Chapter 2.5.7.1 --- Southern blotting --- p.32 / Chapter 2.5.7.2 --- Preparation of α-32P-dCTP labelled GnRH probe --- p.32 / Chapter 2.5.7.3 --- Hybridization --- p.33 / Chapter 2.6 --- Detection of mRNA expression of gonadotropin-releasing hormone receptor (GnRH-R) in normal and neoplastic rat prostates --- p.34 / Chapter 2.6.1 --- Cloning of GnRH-R cDNA and synthesis of its probe --- p.34 / Chapter 2.6.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates by Southern blot --- p.36 / Chapter 2.6.3 --- Detection of GnRH receptor mRNA expression in Dunning tumor --- p.37 / Chapter 2.6.4 --- Detection of the GnRH receptor mRNA expression in AIT tumor by RT-PCR --- p.37 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Detection of mRNA expression of gonadotropin-releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.38 / Chapter 3.1.1 --- Reverse -transcription Polymerase Chain Reaction (RT-PCR) --- p.38 / Chapter 3.1.2 --- Purification of DNA fragments amplified by PCR from the agarose gel --- p.38 / Chapter 3.1.3 --- Subcloning of DNA into vector --- p.39 / Chapter 3.1.4 --- Nucleotide sequencing --- p.39 / Chapter 3.1.5 --- Southern-blot analysis --- p.39 / Chapter 3.2 --- Detection of gonadotropin-releasing hormone receptor mRNA expression in normal and neoplastic rat prostates --- p.40 / Chapter 3.2.1 --- Cloning of gonadotropin-releasing hormone receptor (GnRH) cDNA and synthesis of probe from the normal Noble rat pituitary gland --- p.40 / Chapter 3.2.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates --- p.42 / Chapter 3.2.3 --- Detection of GnRH receptor mRNA expression in rat Dunning tumor by PCR --- p.43 / Chapter 3.2.4 --- Detection of GnRH receptor mRNA expression in AIT tumor --- p.43 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Detection of mRNA expression of gonadotropin-releasing releasing hormone(GnRH) in normal and neoplastic rat prostates --- p.69 / Chapter 4.1.1 --- Expression of GnRH mRNA in normal Nobel rat prostate gland --- p.69 / Chapter 4.1.2 --- Expression of GnRH mRNA in dysplastic Nobel rat prostate --- p.71 / Chapter 4.1.3 --- Expression of GnRH mRNA in androgen-dependent rat Dunning prostatic tumor --- p.72 / Chapter 4.1.4 --- Expression of GnRH mRNA in AIT tumor --- p.74 / Chapter 4.2 --- Detection of GnRH receptor in normal and dysplastic rat prostates --- p.75 / Chapter 4.2.1 --- Negative expression of GnRH receptor in normal and dysplastic Nobel in rat prostates --- p.75 / Chapter 4.2.2 --- Positive expression of GnRH receptor mRNA in rat Dunning tumor --- p.77 / Chapter 4.2.3 --- Negative expression of GnRH receptor mRNA in ALT tumor --- p.78 / Chapter Chapter 5 --- Summary and Conclusions --- p.80 / References --- p.83

Jia, Lin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 124-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Introdução: Apesar dos avanços nas técnicas de diagnóstico e tratamento, o câncer de próstata avançado ainda é uma condição letal. Terapêuticas mais eficazes são necessárias para reduzir as taxas de morbi-mortalidade associadas à doença. A Proteína-78 regulada pela glicose (GRP78), uma proteína de choque térmico envolvida na apresentação de antígenos, foi recentemente descrita como sendo um possível marcador molecular para o câncer de próstata. Ainda mais, a resposta imune a essa proteína mostrou correlação com o desenvolvimento de doença hormônio-independente e com pior sobrevida para a doença. Objetivos: Neste estudo, avaliou-se a hipótese de que a GRP78 poderia ser usada como marcador molecular em câncer de próstata no desenvolvimento de um sistema de receptor-ligante, através do uso da tecnologia de apresentação de fagos. Casuística e métodos: Inicialmente, foram clonados dois peptídeos que apresentam afinidade à proteína regulada pela GRP78 (os peptídeos WIFPWIQL e WDLAWMFRLPVG) no vetor fUSE5, criando-se fagos com capacidade teórica de ligação à mesma proteína. Posteriormente foi testada a capacidade de ligação desses fagos à GRP78 na membrana de células prostáticas malignas em solução, em xeno-tumores in vivo e em metástases ósseas de câncer de próstata humano. Resultados: Demonstrou-se que ambos os fagos se ligam especificamente à GRP78 in vitro, em comparação à proteínas com seqüência semelhante (proteínas de choque térmico 70 e 90) e não semelhante (albumina sérica bovina). Em seguida, mostrou-se que esses fagos se ligam com afinidade pelo menos 30 vezes maior à células de câncer de próstata que o fago controle, e que os fagos são internalizados por essas células. Posteriormente, mostrou-se que os fagos rastrearam xeno-tumores prostáticos quando injetados in vivo num modelo animal de câncer de próstata. Finalmente, mostrou-se que os fagos ligam-se especificamente à GRP78 expressa em metástases ósseas de adenocarcinoma prostático humano. Conclusões: Os fagos criados apresentam capacidade de ligação específica à GRP78 in vitro, em células em suspensão e in vivo. A estratégia e o sistema de receptor-ligante definidos no presente estudo podem ter implicacões relevantes no desenvolvimento de terapias dirigidas para o tratamento do câncer de próstata. / Introduction: Despite the advances in diagnosis and treatment, advanced prostate cancer remains a lethal condition. Improved methods of therapy are needed to reduce the morbidity and mortality rates associated with this disease. The Glucose-regulated protein-78 (GRP78), a stress-responsive heat-shock protein involved in antigen presentation, was recently described as a possible molecular marker for prostate cancer. Moreover, immune response against this protein was shown to have correlation with the development of androgen-independent prostate cancer and shorter overall survival. Objectives: We hipothesized that GRP78 could be used as a molecular marker for prostate cancer in the development of a receptor-ligand system, by using phage display technology. Patients and methods: We initially cloned two GRP78-targeting peptides (WIFPWIQL and WDLAWMFRLPVG) into a fUSE5-based phage. We then tested binding capacity of the phage to GRP78 in vitro, to GRP78 expressed in intact prostate cancer cell membranes, to a prostate cancer xenograft and to human bone metastases. Results: We showed that both phage created bound specifically to GRP78 in vitro, in comparison to related (Heat-shock proteins 70 and 90) and unrelated control proteins (bovine serum albumin). Next, we showed that these phage bound at least 30 times more to prostate cancer cells than the control phage, and were also internalized into these cells. Both GRP78-binding phage showed a strong homing in vivo to a human prostate cancer xenograft in a mouse model. Finally, we showed that both phage bound specifically to GRP78 expressed in human prostate cancer bone metastases. Conclusions: Both phage are capable of binding specifically to GRP78 in vitro, in the context of intact prostate cancer cells and in vivo. The strategy and the ligand-receptor system we have defined in this study may have relevant implications in the development of targeted therapies for the treatment of prostate cancer.

Introdução: Apesar dos avanços nas técnicas de diagnóstico e tratamento, o câncer de próstata avançado ainda é uma condição letal. Terapêuticas mais eficazes são necessárias para reduzir as taxas de morbi-mortalidade associadas à doença. A Proteína-78 regulada pela glicose (GRP78), uma proteína de choque térmico envolvida na apresentação de antígenos, foi recentemente descrita como sendo um possível marcador molecular para o câncer de próstata. Ainda mais, a resposta imune a essa proteína mostrou correlação com o desenvolvimento de doença hormônio-independente e com pior sobrevida para a doença. Objetivos: Neste estudo, avaliou-se a hipótese de que a GRP78 poderia ser usada como marcador molecular em câncer de próstata no desenvolvimento de um sistema de receptor-ligante, através do uso da tecnologia de apresentação de fagos. Casuística e métodos: Inicialmente, foram clonados dois peptídeos que apresentam afinidade à proteína regulada pela GRP78 (os peptídeos WIFPWIQL e WDLAWMFRLPVG) no vetor fUSE5, criando-se fagos com capacidade teórica de ligação à mesma proteína. Posteriormente foi testada a capacidade de ligação desses fagos à GRP78 na membrana de células prostáticas malignas em solução, em xeno-tumores in vivo e em metástases ósseas de câncer de próstata humano. Resultados: Demonstrou-se que ambos os fagos se ligam especificamente à GRP78 in vitro, em comparação à proteínas com seqüência semelhante (proteínas de choque térmico 70 e 90) e não semelhante (albumina sérica bovina). Em seguida, mostrou-se que esses fagos se ligam com afinidade pelo menos 30 vezes maior à células de câncer de próstata que o fago controle, e que os fagos são internalizados por essas células. Posteriormente, mostrou-se que os fagos rastrearam xeno-tumores prostáticos quando injetados in vivo num modelo animal de câncer de próstata. Finalmente, mostrou-se que os fagos ligam-se especificamente à GRP78 expressa em metástases ósseas de adenocarcinoma prostático humano. Conclusões: Os fagos criados apresentam capacidade de ligação específica à GRP78 in vitro, em células em suspensão e in vivo. A estratégia e o sistema de receptor-ligante definidos no presente estudo podem ter implicacões relevantes no desenvolvimento de terapias dirigidas para o tratamento do câncer de próstata. / Introduction: Despite the advances in diagnosis and treatment, advanced prostate cancer remains a lethal condition. Improved methods of therapy are needed to reduce the morbidity and mortality rates associated with this disease. The Glucose-regulated protein-78 (GRP78), a stress-responsive heat-shock protein involved in antigen presentation, was recently described as a possible molecular marker for prostate cancer. Moreover, immune response against this protein was shown to have correlation with the development of androgen-independent prostate cancer and shorter overall survival. Objectives: We hipothesized that GRP78 could be used as a molecular marker for prostate cancer in the development of a receptor-ligand system, by using phage display technology. Patients and methods: We initially cloned two GRP78-targeting peptides (WIFPWIQL and WDLAWMFRLPVG) into a fUSE5-based phage. We then tested binding capacity of the phage to GRP78 in vitro, to GRP78 expressed in intact prostate cancer cell membranes, to a prostate cancer xenograft and to human bone metastases. Results: We showed that both phage created bound specifically to GRP78 in vitro, in comparison to related (Heat-shock proteins 70 and 90) and unrelated control proteins (bovine serum albumin). Next, we showed that these phage bound at least 30 times more to prostate cancer cells than the control phage, and were also internalized into these cells. Both GRP78-binding phage showed a strong homing in vivo to a human prostate cancer xenograft in a mouse model. Finally, we showed that both phage bound specifically to GRP78 expressed in human prostate cancer bone metastases. Conclusions: Both phage are capable of binding specifically to GRP78 in vitro, in the context of intact prostate cancer cells and in vivo. The strategy and the ligand-receptor system we have defined in this study may have relevant implications in the development of targeted therapies for the treatment of prostate cancer.