雖然錯誤調控微小核糖核酸 (miRNA) 引起肝細胞癌 (HCC) 發生發展的生物途徑得到了廣泛的研究，但是對於上游的調控機制卻知之甚少。以往的研究表明，組蛋白甲基化轉移酶 (EZH2) 介導的組蛋白3上27位賴氨酸三甲基化（H3K27me3）是一類通過沉默腫瘤抑制基因而誘發癌症的機制，並且與脫氧核糖核酸 (DNA) 啟動子甲基化機制獨立存在。另一方面，基因抑制也與 H3K27和DNA甲基化相關聯。盡管如此，miRNA沉默機制，特別是在肝癌中，仍然是知之甚少。 / 在這項研究中，我們使用整合全基因組定位和表達分析方法，以探討在肝癌細胞中miRNA表達的表觀遺傳和轉錄控制。通過染色質免疫沉澱偶聯人類啟動子芯片的方法，我們發現在Hep3B和HKCI - 8肝癌細胞中分別有8.4和12.2%的審問miRNA有豐富的H3K27me3。另一方面，在甲基結合域捕捉偶聯芯片實驗中，我們發現在Hep3B和HKCI-8肝癌細胞中分別有15.5和14.7% 的miRNA出現DNA超甲基化。與以往的蛋白質編碼基因結果相同，大多數 H3K27me3豐富的miRNA沒有被檢測到DNA超甲基化，並且反之亦然。 敲除EZH2基因引起H3K27me3水平廣泛下降,並且恢復 H3K27me3抑制的 miRNA表達，而DNA去甲基化劑5-氮雜 -2'-脫氧胞苷 (5-aza-dC) 卻不能重新啟動他們的表達，進一步顯示EZH2基因介導的H3K27me3引發miRNA沉默的機制是獨立存在的。然而，一些過往研究證明有腫瘤抑制功能的miRNA，包括 miR-9-1，miR-9-2和 miR-9-3 被發現同時被 H3K27me3和DNA甲基化調節。我們進一步發現，在肝瘤細胞中，miR-9 特異性調控致癌性的基因結合核因 (NF-κB) 信號通路，並且與配對的非腫瘤肝組織相比，miR-9 的表達在大約一半的原發性肝癌腫瘤（五十九分之三十零）中顯著被壓抑。 / 為了調查在H3K27me3介導的miRNA基因沉默中參與的轉錄因子，我們應用轉錄因子結合位點分析的方法檢查H3K27me3結合蛋白編碼和miRNA基因的調控區域。在包括miR-9亞型的miRNA中，滎陽 1（YY1）的結合位點在這些調控區域中反覆出現並有很高的代表性。定量芯片聯合聚合酶鏈反應結果顯示，在Hep3B細胞中，敲除YY1不僅大大降低了自身的結合力，同時在 miR-9-1，miR-9-2和 miR-9-3 的啟動子中，EZH2基因和H3K27me3結合也大大降低了。尤其重要的是，敲除YY1可以顯著地重新激活他們的表達，表明在肝癌細胞中YY1在EZH2基因介導的的miR-9 表觀遺傳沉默中發揮重要作用。功能研究證明，下調YY1能夠抑制肝癌細胞的增殖，增加細胞凋亡和減少體內的腫瘤生長。定量實時聚合酶鏈反應進一步證實在miR-9 被下調的子集肝癌腫瘤中，有超過85的樣本顯示YY1和EZH2基因同時過量表達，表明我們的研究結果具有臨床相關性。 / 總之，我們完整的分析表明miRNA的調控在肝癌上的獨特表觀遺傳模式。 H3K27me3介導的miRNA沉默可由擁有致癌功能的YY1誘發，它亦可能代表一個可能公認的肝癌癌基因。綜合表觀遺傳和miRNA表達的轉錄控制的結果，能夠提高我們對肝癌發生發展的認識和闡明利用表觀遺傳方法針對性治療肝癌的新的發展方向。 / Although the biological pathways by which mis-regulated microRNAs (miRNAs) contribute to the development of hepatocellular carcinoma (HCC) have been extensively investigated, little is known about the upstream regulatory mechanisms. Previous studies demonstrated that EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation. On the other hand, gene repression can be associated with both H3K27 and DNA methylation. However, the mechanisms underlying miRNA silencing, particularly in HCC, are poorly understood. / In this study, we used an integrated genome-wide location and expression analysis to investigate the epigenetic and transcriptional controls of miRNA expression in HCC cells. Chromatin immunoprecipitation (ChIP) coupled with human promoter microarrays revealed that 8.4 and 12.2% of the interrogated miRNA were enriched with H3K27me3 in Hep3B and HKCI-8 HCC cells, respectively. On the other hand, Methyl-binding domain capture coupled with microarray (MethylCap-chip) uncovered that 15.5 and 14.7% of miRNA were hypermethylated in Hep3B and HKCI-8 HCC cells, respectively. Consistent with previous observation on protein-coding genes, most of the miRNAs enriched with H3K27me3 had no detectable DNA hypermethylation and vice versa. Knockdown of EZH2 decreased global H3K27me3 level and restored expression of the H3K27me3-targeted miRNAs while the DNA demethylating agent 5-aza-2’-deoxycytidine (5-aza-dC) did not reactivate their expression, further suggesting the independence of EZH2-mediated H3K27me3 in miRNA silencing. Nevertheless, a few miRNAs reported to exhibit tumor-suppressive functions including miR-9-1, miR-9-2 and miR-9-3 were found to be regulated by both H3K27me3 and DNA methylation. We further found that miR-9 targeted the oncogenic NF-κB signaling pathway in HCC cells and were significantly repressed in approximately half of the primary HCC tumors (30/59) compared to the paired non-tumor liver tissues. / To investigate the involvement of transcription factors in H3K27me3-mediated gene silencing of miRNAs, the regulatory regions of H3K27me3-bound protein-coding and miRNA genes were submitted to transcription factor binding site analysis. The binding sites for Ying Yang 1 (YY1) were recurrently over-represented in these loci including the miR-9 isoforms. Quantitative ChIP-PCR demonstrated that knockdown of YY1 in Hep3B cells not only significantly reduced its own binding, but also the EZH2 and H3K27me3 promoter occupancy at miR-9-1, miR-9-2 and miR-9-3. Importantly, their expression levels were significantly reactivated by YY1 knockdown, suggesting that YY1 plays part in the EZH2-mediated epigenetic silencing of miR-9 in HCC cells. Functionally, down-regulation of YY1 was shown to inhibit HCC cell proliferation, increase cell apoptosis and reduce tumor growth in vivo. Quantitative RT-PCR further demonstrated that YY1 and EZH2 were concordantly over-expressed in over 85% of the same subset of HCC tumors that exhibited miR-9 down-regulation, demonstrating the clinical relevance of our findings. / In conclusion, our integrated analysis demonstrated differential epigenetic patterns of miRNA regulation in HCC. H3K27me3-mediated silencing of miRNAs may be initiated by YY1, which possesses oncogenic functions and may represent a putative HCC oncogene. The findings of combinatorial epigenetic and transcriptional control of miRNA expression enhance our understanding of hepatocarcinogenesis and shed light on the development of novel epigenetic targeted therapy of HCC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tsang, Pui Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 111-121). / Abstracts also in Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iv / Acknowledgements --- p.vi / table of contents --- p.vii / List of tables --- p.x / List of Figures --- p.xi / Abbreviations --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION4 / Chapter 1.1 --- Hepatocellular carcinoma --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.2 / Chapter 1.2 --- Epigenetic mechanisms --- p.3 / Chapter 1.2.1 --- Epigenetic silencing by DNA hypermethylation --- p.3 / Chapter 1.2.2 --- Epigenetic silencing by Polycomb group protein --- p.5 / Chapter 1.2.3 --- Interplay between H3K27me3 and DNA hypermethylation --- p.7 / Chapter 1.3 --- microRNA --- p.10 / Chapter 1.3.1 --- Transcriptional gene silencing by miRNA --- p.11 / Chapter 1.3.2 --- miRNA and cancers --- p.12 / Chapter 1.3.3 --- miRNA and liver cancer --- p.13 / Chapter 1.4 --- Signal transduction pathway and cancers --- p.14 / Chapter 1.5 --- Aims of study --- p.15 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Cell lines --- p.16 / Chapter 2.2 --- Clinical samples --- p.16 / Chapter 2.3 --- Plasmid DNA transfection --- p.16 / Chapter 2.4 --- Small interfering RNA transfection --- p.17 / Chapter 2.5 --- Extraction of total RNA --- p.19 / Chapter 2.6 --- Western blot analysis --- p.19 / Chapter 2.7 --- Quantitative RT-PCR --- p.20 / Chapter 2.8 --- miRNA Real Time RT-PCR --- p.22 / Chapter 2.9 --- ChIP-chip assay --- p.24 / Chapter 2.10 --- MethylCap-chip --- p.27 / Chapter 2.11 --- miRNA microarray --- p.28 / Chapter 2.12 --- ChIP Assay and Quantitative ChIP-PCR Assay --- p.28 / Chapter 2.13 --- Colony formation assay --- p.33 / Chapter 2.14 --- Cell proliferation assay --- p.33 / Chapter 2.15 --- Annexin V apoptosis assay --- p.34 / Chapter 2.16 --- Cancer 10-pathway reporter array --- p.34 / Chapter 2.16.1 --- Transfection of siEZH2 with 5-aza-dC treatment --- p.34 / Chapter 2.16.2 --- Transfection of siYY1 and pcDNA3-miR9 plasmid --- p.35 / Chapter 2.16.3 --- Luciferase reporter array --- p.35 / Chapter 2.17 --- Animal Studies --- p.36 / Chapter 2.18 --- Statistical Analysis --- p.36 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Occupancy of miRNA genes by epigenetic marks in HCC cells --- p.37 / Chapter 3.1.1 --- Identification of H3K27me3-occupied miRNAs --- p.37 / Chapter 3.1.2 --- Identification of DNA methylation-occupied miRNAs --- p.41 / Chapter 3.1.3 --- Relationship between H3K27me3 and DNA methylation occupancy of miRNAs in HCC cells --- p.45 / Chapter 3.2 --- Regulation of miRNA expression by H3K27me3 and DNA methylation in HCC cells --- p.51 / Chapter 3.3 --- Epigenetic regulation and molecular function of miR-9 in HCC cells --- p.56 / Chapter 3.3.1 --- Confirmation of H3K27me3 and DNA methylation occupancy in miR-9 genes --- p.59 / Chapter 3.3.2 --- Synergistic reactivation of miR-9 upon removal of epigenetic marks --- p.62 / Chapter 3.3.3 --- Effect of miR-9 re-expression on NFKB1 (p50) expression and NF-κB signaling in HCC cells --- p.66 / Chapter 3.4 --- Role of the transcription factor YY1 in the epigenetic regulation of miR-9 --- p.72 / Chapter 3.4.1 --- Identification of transcription factors involved in the regulation of H3K27me3-bound genes --- p.72 / Chapter 3.4.2 --- Occupancy of YY1 on miR-9 in HCC cells --- p.75 / Chapter 3.4.3 --- Effects of YY1 on EZH2/H3K27me3 occupancy and expression of miR-9 --- p.78 / Chapter 3.4.4 --- Effects of YY1 on p50/p65 expression and NF-κB signaling in HCC cells --- p.81 / Chapter 3.5 --- Functional significance of YY1 in HCC --- p.84 / Chapter 3.5.1 --- Effect of YY1 on HCC cell growth --- p.84 / Chapter 3.5.2 --- Effect of YY1 on HCC cell apoptosis --- p.87 / Chapter 3.5.3 --- Effect of YY1 on HCC cell growth in vivo --- p.90 / Chapter 3.5.4 --- Expressions of YY1, EZH2 and miR-9 on clinical HCC samples --- p.92 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- Independence of EHZ2-mediated H3K27me3 and DNA methylation --- p.97 / Chapter 4.2 --- Concordant H3K27 and DNA methylation-mediated silencing of miR-9 --- p.101 / Chapter 4.3 --- Ectopic expression of miR-9 inhibits NF-kB signaling in HCC cells --- p.102 / Chapter 4.4 --- YY1 is involved in the regulation of H3K27me3-bound genes --- p.103 / Chapter 4.5 --- Knockdown of YY1 inhibits NF-kB signaling in HCC --- p.105 / Chapter 4.6 --- Clinical relevance and therapeutic significance of miR-9 silencing by YY1-mediated recruitment of EZH2 --- p.106 / Chapter 4.7 --- Limitations and future studies --- p.109 / REFERENCES --- p.111 / PUBLICATION --- p.122

Protection of business competition - agreements interfering with competition Agreements interfering with competition are a significant threat to proper functioning of the competition. Leniency programmes are a powerful tool for the purpose of detection and termination of cartels. This thesis focuses mainly on the regulation contained in the Czech national law. The Community law can not be ignored though as the national law is closely connected therewith. The first part of this thesis contains mainly a basic definition of agreements interfering with competition, their division according to their content as well as division to horizontal and vertical agreements which is important to assess agreements with an insignificant impact on the competition. When assessing the influence of an agreement it is also necessary to take legal and block exemptions into account. The most important cartel assessed by the Office for the Protection of Competition so far is an agreement on the gas insulated switchgear market. For this agreement having a form of bid rigging the Office imposed the highest fine in its history. But this case also demonstrates a frequent problem of significant delays in the court proceedings, which are often initiated by legal actions of undertakings against decisions of the Office for the...

Ng, Wing Chi Linda. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-142). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.iv / Publications --- p.v / Abbreviations --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Stem Cells --- p.1 / Chapter 1.1.1 --- Definition of Stem Cells --- p.1 / Chapter 1.1.2 --- Different Origins of Stem Cells --- p.2 / Chapter 1.1.3 --- Challenges and Importance of Stem Cell Research --- p.5 / Chapter 1.2 --- Adult Mesenchymal Stem Cells --- p.7 / Chapter 1.2.1 --- Characteristics of Adult Mesenchymal Stem Cells --- p.7 / Chapter 1.2.2 --- Adipose Tissue as an Alternate Source of MSCs --- p.8 / Chapter 1.2.3 --- Adipose Tissue Versus Bone Marrow as a Source of MSCs --- p.10 / Chapter 1.3 --- Adipose Tissue-derived Stem Cells (ATSCs) --- p.11 / Chapter 1.3.1 --- Cell Surface Marker Characteristic of ATSCs --- p.11 / Chapter 1.3.2 --- Global Gene Expression Profile of ATSCs --- p.14 / Chapter 1.3.3 --- Immunomodulatory Effect of ATSCs --- p.15 / Chapter 1.3.4 --- Proliferation Capacity of ATSCs --- p.17 / Chapter 1.3.5 --- Multilineage Differentiation of ATSCs --- p.18 / Chapter 1.3.5.1 --- Differentiation Capability of ATSCs : Adipogenesis --- p.18 / Chapter 1.3.5.2 --- Osteogenesis --- p.19 / Chapter 1.3.5.3 --- Skeletal and Smooth Muscle Myogenesis --- p.21 / Chapter 1.3.5.4 --- Cardiomyogenesis --- p.23 / Chapter 1.3.5.5 --- Chondrogenesis --- p.24 / Chapter 1.3.5.6 --- Neurogenesis --- p.27 / Chapter 1.4 --- Signaling Pathways in Stem Cells --- p.31 / Chapter 1.4.1 --- Wnt Signaling --- p.31 / Chapter 1.4.2 --- Notch Signaling --- p.33 / Chapter 1.4.3 --- Signaling Pathway of the TGF-β Superfamily --- p.34 / Chapter 1.5 --- Pathways Controlling Chondrogenesis --- p.36 / Chapter 1.6 --- MicroRNA --- p.39 / Chapter 1.6.1 --- MicroRNA - A Novel Gene Regulator --- p.39 / Chapter 1.6.2 --- Biogenesis of MicroRNAs --- p.40 / Chapter 1.6.3 --- Post-transcriptional Repression by MicroRNAs --- p.43 / Chapter 1.6.4 --- Role of MicroRNAs in Development --- p.45 / Chapter 1.6.5 --- MicroRNAs in Stem Cell Differentiation --- p.46 / Chapter 1.6.5.1 --- MicroRNA Expression Profile in ESCs --- p.46 / Chapter 1.6.5.2 --- Lineage Differentiation --- p.47 / Chapter 1.7 --- Project Aims --- p.52 / Chapter 1.8 --- Significance of Study --- p.53 / Chapter Chapter 2 --- Materials and Methods --- p.54 / Chapter 2.1 --- Sample Collection --- p.54 / Chapter 2.2 --- Isolation and Culture of ATSCs --- p.54 / Chapter 2.3 --- Measurement of Cell Growth --- p.55 / Chapter 2.4 --- Effect of Estrogen Treatment on ATSC Proliferation --- p.55 / Chapter 2.5 --- Multilineage Differentiation of ATSCs --- p.55 / Chapter 2.5.1 --- Chondrogenic Differentiation --- p.56 / Chapter 2.5.2 --- Neural Differentiation --- p.56 / Chapter 2.6 --- Immunocytochemical Analysis of Surface Markers and Lineage Specific Markers --- p.57 / Chapter 2.7 --- Alcian Blue Staining --- p.58 / Chapter 2.8 --- RNA Extraction --- p.58 / Chapter 2.9 --- Reverse Transcription --- p.59 / Chapter 2.10 --- Quantitative Real-time Polymerase Chain Reaction --- p.59 / Chapter 2.11 --- Statistical Analysis of Real-time PCR Data --- p.61 / Chapter 2.12 --- MicroRNA Profiling --- p.61 / Chapter 2.12.1 --- Reverse Transcription --- p.62 / Chapter 2.12.2 --- Quantitative Real-time Polymerase Chain Reaction --- p.62 / Chapter 2.13 --- mRNA Target Prediction of MicroRNA --- p.63 / Chapter 2.14 --- MicroRNA Knockdown Assay --- p.63 / Chapter 2.15 --- MicroRNA Over-expression Assay --- p.64 / Chapter 2.15.1 --- Vector Amplification --- p.64 / Chapter 2.15.1.1 --- Transformation --- p.64 / Chapter 2.15.1.2 --- Purification of Plasmid DNA --- p.65 / Chapter 2.15.1.3 --- Confirmation of Construct Insertion --- p.66 / Chapter 2.15.2 --- Transfection of Plasmid and Establishment of MicroRNA Precursor Expressing Cell Lines --- p.66 / Chapter 2.16 --- Gene Expression Microarry --- p.67 / Chapter 2.16.1 --- Preparation of Amplification and Labeling Reaction --- p.67 / Chapter 2.16.2 --- Purification of the Labeled/Amplified RNA --- p.68 / Chapter 2.16.3 --- RNA Fragmentation --- p.68 / Chapter 2.16.4 --- Hybridization --- p.69 / Chapter 2.16.5 --- Array Washing and Scanning --- p.69 / Chapter 2.16.6 --- Statistical Analysis of Microarray Data --- p.69 / Chapter CHAPTER 3 --- RESULTS --- p.71 / Chapter 3.1 --- Isolation and Characterization of ATSCs --- p.71 / Chapter 3.2 --- ATSCs Exhibited Multilineage Differentiation --- p.75 / Chapter 3.2.1 --- Chondrogenic Differentiation --- p.75 / Chapter 3.2.2 --- Expression of Chondrogenic Markers --- p.76 / Chapter 3.2.3 --- Neural Differentiation --- p.80 / Chapter 3.2.4 --- Expression of Neural Markers --- p.83 / Chapter 3.3 --- Effect of Donor's Reproductive Status on the Proliferation and Differentiation Capacity of ATSCs --- p.83 / Chapter 3.3.1 --- Expression of Stem Cell Makers --- p.86 / Chapter 3.3.2 --- Cell Proliferation Assay --- p.86 / Chapter 3.3.3 --- Differentiation Capacity of ATSCs --- p.89 / Chapter 3.4 --- Effect of E2 Treatment on the Proliferation Rate of ATSCs --- p.89 / Chapter 3.5 --- MicroRNA --- p.91 / Chapter 3.5.1 --- MicroRNA Expression Profile of Undifferentiated and Chondrogenic Differentiated ATSCs --- p.91 / Chapter 3.5.2 --- Clustering Analysis Identified MicroRNAs Segregate with ATSCs --- p.91 / Chapter 3.5.3 --- Identification of Differentially Expressed MicroRNAs in Chondrogenic-induced ATSCs --- p.95 / Chapter 3.5.4 --- mRNA Target Prediction for miR-199a --- p.97 / Chapter 3.6 --- Correlating MicroRNA Expression and mRNA Levels: Clues to MicroRNA Function --- p.97 / Chapter 3.6.1 --- Effect ofmiR-199a RNAi in Phenotypic Changes of Chondrogenic-induced ATSCs --- p.97 / Chapter 3.6.2 --- Identification of Potential Target Genes by Microarray Analysis of ATSCs with miR-199a Over-expression and Knockdown --- p.102 / Chapter CHAPTER 4 --- DISCUSSION --- p.104 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.115 / APPENDICES --- p.117 / REFERENCES --- p.120

Prostate cancer is one of the leading causes of death among males in the United States. In this study, we hypothesized that an activated PKC-iota-dependent anti-apoptotic pathway, drives the cell cycle proliferation and survival of prostate cancer cells. We investigated the role of atypical PKC-iota (PKC-ι) in androgen- independent prostate DU-145 carcinoma, androgen-dependent prostate LNCaP carcinoma compared to transformed non-malignant prostate RWPE-1 cells. Western blotting and immunoprecipitations demonstrated that PKC-ι is associated with cyclin-dependent activating kinase (CAK/Cdk7) in androgen-dependent, RWPE-1 and LNCaP cells but not in androgen-independent DU-145 cells. Treatment of prostate RWPE-1 cells with PKC-ι silencing RNA (siRNA) decreased cell proliferation, cell cycle accumulation at G2/M phase and decreased phosphorylation of Cdk7 and cdk2. In addition, PKC-ι siRNA treatment provoked a decrease in phosphorylation of Bad and increased Bad/Bcl-xL heterodimerization, leading to cell apoptosis. In DU-145 cells, PKC-ι is anti-apoptotic and still required for cell survival. Treatment with PKC-ι siRNA blocked an increase in cell number, and inhibited G1/S transition. In addition to cell cycle arrest, both RWPE-1 cells and DU-145 cells underwent apoptosis via mitochondria dysfunction and activating apoptosis cascades such as release of cytochrome c, activation of caspase-7, and poly-(ADP-ribose) polymerase (PARP) cleavage. Mechanistic pathways involving aPKCs in the NF-κB survival pathway were established using pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα). Results demonstrated that RWPE-1 cells and DU-145 cells are insensitive to TNFα whereas LNCaP cells are sensitive to TNFα treatment and undergo apoptosis. In DU-145 cells, TNFα induced PKC-ι activation of IκB kinase, IKKα/ß, while in RWPE-1 cells, PKC-ζ activates IKKα/ß. Both RWPE-1 and DU-145 show degradation of IκBα allowing NF-κB/p65 translocation to the nucleus. In LNCaP cells, the upstream kinase activation IKKα/ß was not observed, although there have been reports that LNCaP cells weakly activate IKKα and have NF-κB activation. In vivo kinase assay demonstrates that PKC-ι is the substrate of IKKα/ß. A putative PKC-ι inhibitor (ICA-1) inhibited activation of IKKα/ß in vivo. Hence, PKC-ι is an antiapoptotic protein and this suggests that anti-PKC-ι therapy may be a viable option for prostate carcinoma cells.

AIDS is caused by a lentivirus, HIV-1. In addition to antiretroviral drugs that are currently in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed as alternative therapies. Most of these therapies target HIV RNA/proteins, which are subject to high rate of mutation, resulting in escape mutants. Viral entry is mediated by CCR5 co-receptor in most routes of transmission. To downregulate CCR5 as a gene therapy approach, we targeted seven unique sites within the CCR5 mRNA by a multimeric hammerhead ribozyme, Rz1-7. Hammerhead ribozyme is a small RNA that cleaves a target RNA upon binding to it. Expressing the Rz1-7 from HIV-1- and MSCV-based vectors in otherwise susceptible cells inhibited replication of a CCR5-tropic strain of HIV-1 by 99-100%. The Rz1-7 will be tested for inhibition of HIV-1 replication in the CD4+ T-lymphoid and myeloid progeny of transduced human CD34+ hematopoietic progenitor stem cells. It may be preferable to interfere HIV-1 life cycle at the DNA level since a one-time inactivation might suffice to confer a complete and permanent inhibition of virus replication in the gene modified cells and their progeny. This is what other strategies that target the HIV-1 RNA/protein can hardly offer. For this purpose, group II introns, which are able to splice out and get incorporated into a specific DNA sequence, can be designed/modified to gain novel DNA targeting specificities. As a novel approach, we have examined whether insertion of a modified intron into an infectious HIV-1 clone at two sites within the integrase domain of HIV-1 pol gene could inhibit virus replication. Intron insertion into the HIV-1 clone was induced and mammalian cells were transfected with intron-inserted HIV-1 clones. Although similar amounts of HIV-1 RNA, protein, and progeny virus were produced from the clones as from wild-type HIV-1 provirus DNA, in the absence of a functional integrase, the HIV-1 reverse-transcribed DNA failed to integrate and virus replication was aborted. These results demonstrate that modified group II introns can confer complete inhibition of virus replication at the level of second round of infection. We are now developing vectors to assess whether intron insertion can take place in mammalian cells.

AIDS is caused by a lentivirus, HIV-1. In addition to antiretroviral drugs that are currently in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed as alternative therapies. Most of these therapies target HIV RNA/proteins, which are subject to high rate of mutation, resulting in escape mutants. Viral entry is mediated by CCR5 co-receptor in most routes of transmission. To downregulate CCR5 as a gene therapy approach, we targeted seven unique sites within the CCR5 mRNA by a multimeric hammerhead ribozyme, Rz1-7. Hammerhead ribozyme is a small RNA that cleaves a target RNA upon binding to it. Expressing the Rz1-7 from HIV-1- and MSCV-based vectors in otherwise susceptible cells inhibited replication of a CCR5-tropic strain of HIV-1 by 99-100%. The Rz1-7 will be tested for inhibition of HIV-1 replication in the CD4+ T-lymphoid and myeloid progeny of transduced human CD34+ hematopoietic progenitor stem cells. It may be preferable to interfere HIV-1 life cycle at the DNA level since a one-time inactivation might suffice to confer a complete and permanent inhibition of virus replication in the gene modified cells and their progeny. This is what other strategies that target the HIV-1 RNA/protein can hardly offer. For this purpose, group II introns, which are able to splice out and get incorporated into a specific DNA sequence, can be designed/modified to gain novel DNA targeting specificities. As a novel approach, we have examined whether insertion of a modified intron into an infectious HIV-1 clone at two sites within the integrase domain of HIV-1 pol gene could inhibit virus replication. Intron insertion into the HIV-1 clone was induced and mammalian cells were transfected with intron-inserted HIV-1 clones. Although similar amounts of HIV-1 RNA, protein, and progeny virus were produced from the clones as from wild-type HIV-1 provirus DNA, in the absence of a functional integrase, the HIV-1 reverse-transcribed DNA failed to integrate and virus replication was aborted. These results demonstrate that modified group II introns can confer complete inhibition of virus replication at the level of second round of infection. We are now developing vectors to assess whether intron insertion can take place in mammalian cells.

The therapeutic potential of a novel multi-targeted small-interfering RNA (siRNA) was investigated in human breast cancer cells. Previous studies had identified an siRNA that specifically and potently inhibited expression of thymidylate synthase (TS) by directly targeting human TS mRNA. TS is a folate-dependent enzyme that catalyzes the key reaction involved in synthesizing nucleotide precursors for DNA biosynthesis, and as such, it plays a critical role in maintaining cell growth. The goal of this thesis was to design and develop a novel siRNA molecule that targeted TS mRNA as well as a cellular mRNA that encodes a different cellular protein involved in cancer cell growth and proliferation, such as a member of the ErbB family. Gene sequence analysis was performed and identified an overlapping sequence between TS and ErbB2 mRNAs. An siRNA duplex was then designed to simultaneously target human TS and ErbB2 mRNA. Transfection of the multi-targeted siRNA (TS1M17) revealed that both ErbB2 and TS proteins were significantly suppressed in a time and dose-dependent manner in ErbB2-overexpressing human breast cancer SKBR3 cells. The corresponding mRNA levels, as determined by RT-PCR, were also decreased. Protein levels of other ErbB family members, including ErbB1 and ErbB3, remained unchanged with siRNA treatment. An ErbB2-specific siRNA (B2450) inhibited ErbB2, but had no effect on TS expression demonstrating the specificity of the multi-targeted siRNA against both TS and ErbB2. Mismatched (TS1-Mismatch) and control (GL2) siRNAs had no inhibitory effects on expression of the two target proteins. Suppression of activated ErbB2, as determined by expression of phosphorylated ErbB2 protein, was observed with transfection of TS1M17 siRNA. In addition, the expression of downstream signaling proteins, such as phosphorylated mitogen activated protein kinase (p-MAPK), p27Kip1, p21Cip1, cyclin D1, and survivin were significantly changed. In contrast, control siRNAs did not exert any inhibitory effects on downstream signaling. Taken together, these findings suggest that TS1M17 siRNA inhibits signaling of the ErbB2 pathway. The effect of TS1M17 siRNA on cytotoxicity was analyzed by WST-1 assay. Upon transfection into SKBR3 cells, the TS1M17 siRNA significantly suppressed cell proliferation with an IC50 value of 0.65 nM, which is 154-fold more potent than ErbB2- and TS-specific siRNAs. This study suggests that targeting expression of ErbB2 and TS, two key proteins involved in distinct and critical pathways for cancer growth and proliferation, with a single siRNA molecule may provide a novel approach for cancer chemotherapy.

published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy