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Leung Chin-lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 133-164). / Abstracts in English and Chinese. / Abstract --- p.iv / 摘要 --- p.vi / List of abbreviation --- p.viii / Chapter Chapter 1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1 --- A Health Burden --- p.1 / Chapter 1.2 --- Pathology --- p.3 / Chapter 1.3 --- Epidemiology --- p.7 / Chapter 1.3.1 --- Global HCC distribution --- p.7 / Chapter 1.3.2 --- Age and Gender --- p.10 / Chapter 1.4 --- Risk Factors of HCC --- p.12 / Chapter 1.4.1 --- Hepatitis B virus (HBV) --- p.13 / Chapter 1.4.1.1 --- Chronic HBV infection --- p.13 / Chapter 1.4.1.2 --- Role of HBV in hepatocarcinogenesis --- p.16 / Chapter 1.4.1.2 a) --- Direct Oncogenesis --- p.16 / Chapter 1.4.1.2 b) --- Indirect Oncogenesis --- p.17 / Chapter 1.4.2 --- Hepatitis C virus (HCV) --- p.23 / Chapter 1.4.2.1 --- Chronic HCV infection --- p.23 / Chapter 1.4.2.2 --- Role of HCV in hepatocarcinogenesis --- p.23 / Chapter 1.4.3 --- Chemicals as liver carcinogens --- p.27 / Chapter 1.4.3.1 --- Aflatoxin Bi (AFB1) --- p.28 / Chapter 1.4.3.2 --- Vinyl chloride --- p.29 / Chapter 1.4.3.3 --- Alcoholic beverages --- p.29 / Chapter 1.4.4 --- Inborn Errors in Metabolisms --- p.30 / Chapter 1.4.4.1 --- Hereditary tyrosinemia --- p.30 / Chapter 1.4.4.2 --- Hereditary haemochromatosis --- p.30 / Chapter 1.4.4.3 --- α1-antitrypsin deficiency --- p.31 / Chapter 1.4.5 --- Liver lesions --- p.32 / Chapter 1.5 --- Genetic alterations in HCC --- p.33 / Chapter Chapter 2 --- Rationale of the study --- p.39 / Chapter Chapter 3 --- LOH study on 8p in HCC --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.1.1 --- "Knudson's ""two-hit"" model and LOH" --- p.48 / Chapter 3.1.2 --- Microsatellite DNA and LOH study --- p.49 / Chapter 3.2 --- Materials and Methods --- p.51 / Chapter 3.2.1 --- Patients and Specimens --- p.51 / Chapter 3.2.1.1 --- Genomic DNA extraction from liver tissues --- p.53 / Chapter 3.2.1.2 --- Genomic DNA extraction from buffy coat --- p.55 / Chapter 3.3 --- LOH study on 8p in HCC --- p.57 / Chapter 3.3.1 --- Microsatellite markers --- p.57 / Chapter 3.3.2 --- 5-end labeling --- p.60 / Chapter 3.3.3 --- Amplification of microsatellite DNA --- p.60 / Chapter 3.3.4 --- Denaturing polyacrylamide gel electrophoresis --- p.61 / Chapter 3.3.5 --- Detection of LOH --- p.62 / Chapter 3.4 --- Results --- p.63 / Chapter 3.4.1 --- LOH status of 52 HCC cases --- p.63 / Chapter 3.4.2 --- Clinicopathological correlation --- p.67 / Chapter 3.4.3 --- Delineation of common deletion region (CDR) --- p.67 / Chapter 3.4.4 --- Common deletion region of interest --- p.77 / Chapter Chapter 4 --- Study on LZTS1 --- p.83 / Chapter 4.1 --- Introduction 一 LZTS1 --- p.83 / Chapter 4.2 --- Mutation analysis of LZTS1 in HCC --- p.87 / Chapter 4.2.1 --- Materials and Methods --- p.87 / Chapter 4.2.1.1 --- Patients and HCC cell lines --- p.87 / Chapter 4.2.1.2 --- Genomic DNA extraction from HCC cell lines --- p.87 / Chapter 4.2.1.3 --- Amplification of exons of LZTS1 --- p.89 / Chapter 4.2.1.3a) --- Primer pairs --- p.89 / Chapter 4.2.1.3b) --- PCR conditions --- p.90 / Chapter 4.2.1.4 --- Purification of PCR products --- p.93 / Chapter 4.2.1.5 --- Cycle sequencing reaction --- p.94 / Chapter 4.2.1.6 --- Purification of cycle sequencing reaction product --- p.94 / Chapter 4.2.1.7 --- Sequence analysis by automated sequencer --- p.95 / Chapter 4.2.1.8 --- Search for sequence variants of LZTS1 --- p.96 / Chapter 4.2.2 --- Results --- p.97 / Chapter 4.3 --- Expression analysis of LZTS1 in HCC with preliminary results --- p.103 / Chapter 4.3.1 --- Materials and Methods --- p.103 / Chapter 4.3.1.1 --- Patients and Specimens --- p.103 / Chapter 4.3.1.2 --- Total RNA extraction --- p.103 / Chapter 4.3.1.3 --- Reverse transcription --- p.104 / Chapter 4.3.1.4 --- Semi-quantitative PCR --- p.105 / Chapter 4.3.1.4a) --- Primer pairs --- p.105 / Chapter 4.3.1.4b) --- PCR conditions --- p.106 / Chapter 4.3.2 --- Results --- p.109 / Chapter Chapter 5 --- Discussion --- p.111 / Chapter 5.1 --- LOH study on 8p in HCC --- p.111 / Chapter 5.2 --- Study on LZTS1 in HCC --- p.125 / Chapter 5.2.1 --- Mutation analysis of LZTS1 --- p.125 / Chapter 5.2.2 --- Expression analysis of LZTS1 --- p.129 / Chapter 5.3 --- Future Study --- p.132 / References --- p.133

抑制腫瘤的基因（又稱抑癌基因）之表達失活，是導致癌變的重要機制之一。除了基因突變之外，越來越多研究證明抑癌基因的關閉轉錄，是由於抑癌基因啟動子區的CpG島甲基化所致。本論文的研究確定了兩個候選抑癌基因ADAMTS8和TUSC14，在多種腫瘤細胞株中經常因動子區的CpG島甲基化而下調或停止表達，這有別於它們在正常組織中廣泛表達的情況。沉默細胞株在脫氧核糖核酸甲基轉移酶的抑製劑5-氮-2'-脫氧胞苷(5-aza-2′-deoxycytidine; Aza) 或與組蛋白去乙酰化酶抑製劑曲古抑菌素A (trichostatin A, TSA)的去甲基化作用下，能恢復這兩個抑癌基因的表達，因而證明了啟動子甲基化是直接導致其表達下調及沉默的機制。 / 論文的第一部分，主要調查ADAMTS8啟動子區在原發腫瘤樣本被甲基化的比率，並研究其腫瘤抑制功能。含血小板凝血酶敏感蛋白基序的解整聯蛋白金屬蛋白酶 (ADAMTSs) ，在各種癌症中的表達異常已有報導。然而，它們在腫瘤的職能作用仍然模糊不清。本研究發現，異位表達ADAMTS8誘導細胞凋亡，因而顯著抑制腫瘤細胞克隆形成的能力,。這些都突顯其抑制腫瘤的功能。此外，作為分泌蛋白酶的ADAMTS8，能夠透過減少表皮生長因子受體(EGFR) 蛋白的磷酸化，抑制EGFR / MEK / ERK信號通路，並進一步破壞肌動蛋白應力纖維的組織，抑制腫瘤細胞的遷移性。 / 論文的第二部分，集中於研究一個未知功能的基因TUSC14，這基因的蛋白質編碼具有氨基末端蛋白質相互作用域 (BTB/POZ domain)及C₂H₂乙炔鋅指結構。TUSC14的異位表達能抑制腫瘤細胞克隆的形成，但這種抑制作用會在删除蛋白中的BTB/POZ或C₂H₂乙炔鋅指結構功能域後消失。因此證實了TUSC14蛋白同時需要BTB / POZ和C₂H₂乙炔鋅指結構兩個功能域來抑制腫瘤生長。此外，TUSC14具有抑制NF-kB轉錄的功能，其功能不但依賴於组蛋白去乙酰基酶(HDAC)，並且與c-MYC和cIAP-2等NF-κB靶基因下調表達相關。TUSC14的抑癌功能，包括抑制腫瘤生長與增加細胞凋亡，與其減少c-MYC及抗凋亡基因cIAP-2的表達，效果一致。進一步的分析發現，TUSC14與HDAC1和P65於蛋白質複雜免疫共沉澱實驗中，有物理相互作用。此外，染色質免疫沉澱實驗顯示TUSC14透過與c-MYC和cIAP-2的相互作用，抑制其基因啟動子區的轉錄功能。結果表明，TUSC14是通過招募HDAC至NF-κB靶基因的啟動子區這機制，來抑制NF-kB靶基因的轉錄，以達至抑制癌細胞生長和誘導癌細胞凋亡的效果。因此，TUSC14的沉默是破壞癌細胞中NF-kB信號通路負調控(negative regulation)的重要因素。 / 綜上所述，本研究鑒定了兩個在多種腫瘤細胞因表觀遺傳沉默效應而表達下調或沉默的抑癌基因ADAMTS8和TUSC14，並證實它們具有抑癌功能。 / Inactivation of tumor suppressor genes (TSGs) is one of the critical mechanisms leading to carcinogenesis. Apart from genetic mutations, a growing number of TSG has been shown to be silenced through promoter CpG methylation. In this thesis, we identified two candidate TSGs: ADAMTS8 and TUSC14 that are frequently downregulated or silenced in multiple carcinoma cell lines by promoter methylation while broadly expressed in normal tissues. Expression of these two genes was restored after treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) or in combination with a histone deacetylase inhibitor trichostatin A (TSA), suggesting promoter-methylation directly contributes to their silencing. / In the first part of the thesis, prevalence silencing of ADAMTS8 was detected in primary tumor samples. Expression of many disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) was reported to be dysregulated in various cancers. However, their functional roles in tumorigenesis remain obscure. This study revealed that ectopic expression of ADAMTS8 markedly inhibits tumor cell clonogenicity by inducing apoptosis, underscoring its function as a tumor suppressor. Furthermore, ADAMTS8, as a secreted protease, inhibits EGFR/MEK/ERK signaling pathway by reducing their phosphorylation, further resulting in the disruption of actin stress fiber organization and suppression of tumor cell motility. / The second part of the thesis focused on a novel gene TUSC14 which encodes a protein with BTB/POZ domain and C₂H₂zinc-fingers. Ectopic expression of TUSC14 suppresses colony formation of cancer cells but this inhibitory effect is abolished with the deletion of BTB/POZ domain or C₂H₂ zinc-fingers. This suggested that both BTB/POZ domain and C₂H₂ zinc-fingers are required for inhibiting tumor cell clonogenecity. In addition, TUSC14 functions as a transcriptional repressor of NF-kB pathway that is dependent on HDAC. Suppression of NF-κB transcriptional activity by TUSC14 expression correlates with the downregulation of NF-κB target genes including c-MYC and cIAP-2. Reduction of c-MYC and anti-apoptotic cIAP-2 agrees well with the consequent growth suppression and enhanced apoptosis following the ectopic expression of TUSC14. Further analyses showed TUSC14 physically interacts with HDAC1 and p65 via co-immunoprecipitation assay. Preliminary ChIP assay showed that TUSC14 associates with gene promoters of c-MYC and cIAP-2 for their transcription repressions. These results revealed that TUSC14 represses NF-kB activity through recruiting HDAC to the NF-kB target genes; and transcription repression of NF-kB represents a mechanism for TUSC14 to mediate its growth inhibitory and apoptosis-inducing effects in cancer. Hence, silencing of TUSC14 contributes to the lost of negative regulation on NF-kB signaling in cancer. / In summary, this study demonstrated that ADAMTS8 and TUSC14 are functional tumor suppressors that are epigenetically silenced in multiple tumors. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Choi, Ching Gee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 140-153). / Abstract also in Chinese. / Abstract --- p.i / Chinese abstract --- p.iv / AcknowledgEments --- p.vii / List of Figures --- p.ix / List of Tables --- p.xi / LIST OF ABBREVIATIONS --- p.xii / List of PUBLICATIONs --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of cancer epigenetics --- p.1 / Chapter 1.2 --- Epigenetic events --- p.2 / Chapter 1.2.1 --- DNA methylation --- p.2 / Chapter 1.2.2 --- Histone modifications --- p.5 / Chapter 1.2.3 --- The interdependence of DNA methylation and histone modifications --- p.8 / Chapter 1.3 --- Epigenetic alterations in cancer --- p.9 / Chapter 1.3.1 --- Genome-wide DNA hypomethylation --- p.9 / Chapter 1.3.2 --- CpG island promoter hypermethylation silencing of tumour suppressor genes in tumorigenesis --- p.10 / Chapter 1.3.3 --- Aberrations of histone modifications --- p.11 / Chapter 1.4 --- Causes for epigenetic deregulation in cancer --- p.14 / Chapter 1.5 --- The interplay of genetic and epigenetic aberration in cancer progression --- p.21 / Chapter 1.6 --- Epigenetic inactivation of tumor suppressor genes in cancer --- p.23 / Chapter 1.7 --- Clinical implications of epigenetic research --- p.27 / Chapter 1.7.1 --- Epigenetic modifications as biomarker for cancer diagnosis --- p.27 / Chapter 1.7.2 --- Targeting epigenetic modifications as therapeutics towards cancers --- p.29 / Chapter 1.8 --- Roles of ADAMTS proteins in cancer --- p.32 / Chapter 1.8.1 --- Introduction on ADAMTS metalloproteases --- p.32 / Chapter 1.8.2 --- Deregulation of ADAMTS protein in cancer --- p.34 / Chapter 1.9 --- Roles of BTB/POZ-ZF family of transcription factors in cancer --- p.36 / Chapter 1.9.1 --- Introduction on BTB/POZ-ZF Family --- p.36 / Chapter 1.9.2 --- BTB/POZ-ZF functions as transcription repressors --- p.37 / Chapter 1.9.3 --- Many BTB/POZ-ZF proteins are important players in tumorigenesis --- p.39 / Chapter 1.9.4 --- The role of BTB/POZ-ZF in tumor initiation and progression --- p.40 / Chapter CHAPTER 2 --- Aims of this study --- p.44 / Chapter CHAPTER 3 --- General Methodology --- p.46 / Chapter 3.1 --- Cell Culture --- p.46 / Chapter 3.1.1 --- Growth and maintenance of cells --- p.46 / Chapter 3.1.2 --- Mammalian cell transfection --- p.46 / Chapter 3.1.3 --- Drug and stress treatments --- p.47 / Chapter 3.2 --- DNA and RNA extraction --- p.47 / Chapter 3.3 --- Semi-quantitative RT-PCR and Real-time PCR --- p.48 / Chapter 3.4 --- CpG island and Transcription factor binding sites analysis --- p.49 / Chapter 3.5 --- Methylation-specific PCR (MSP) and Bisulfite genomic sequencing (BGS) --- p.49 / Chapter 3.6 --- Bacterial transformation and Plasmid extraction --- p.50 / Chapter 3.6.1 --- Heat-shock transformation --- p.50 / Chapter 3.6.2 --- Mini-scale preparation of plasmid DNA --- p.51 / Chapter 3.6.3 --- Preparation of endotoxin-free plasmids --- p.52 / Chapter 3.7 --- DNA cycle sequencing --- p.52 / Chapter 3.8 --- Indirect immunofluorescence for subcellular localization study --- p.54 / Chapter 3.9 --- Colony formation assay --- p.54 / Chapter 3.10 --- Cell cycle analysis --- p.55 / Chapter 3.11 --- Apoptosis assay --- p.56 / Chapter 3.12 --- Co-immunoprecipitation and Western blot --- p.56 / Chapter 3.13 --- Chromatin immunoprecipitation (ChIP) --- p.58 / Chapter 3.14 --- Dual Firefly and Renilla luciferase reporter gene assay --- p.59 / Chapter 3.15 --- Statistical analysis --- p.60 / Chapter CHAPTER 4: --- Characterization of the Tumor Suppressive Functions of ADAMTS8 --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Materials and Methods --- p.64 / Chapter 4.2.1 --- Tumor samples --- p.64 / Chapter 4.2.2 --- Expression of ADAMTS8 --- p.64 / Chapter 4.2.3 --- Immunofluorescence staining of ADAMTS8 --- p.64 / Chapter 4.2.4 --- Detection of secreted ADAMTS8 in culture medium --- p.65 / Chapter 4.2.5 --- Collection of conditioned medium and Western Blotting --- p.66 / Chapter 4.2.6 --- Wound healing assay --- p.66 / Chapter 4.3 --- Result and Discussion --- p.69 / Chapter 4.3.1 --- Frequent ADAMTS8 methylation in primary carcinomas --- p.69 / Chapter 4.3.2 --- ADAMTS8 is a secreted protease --- p.70 / Chapter 4.3.3 --- ADAMTS8 inhibits phosphorylation of pEGFR --- p.73 / Chapter 4.3.4 --- ADAMTS8 suppresses cell migration --- p.77 / Chapter 4.4 --- Summary --- p.81 / Chapter CHAPTER 5: --- Epigenetic Alterations of TUSC14 Gene in multiple carcinomas --- p.83 / Chapter 5.1 --- Introduction --- p.83 / Chapter 5.2 --- Materials and Methods --- p.84 / Chapter 5.2.1 --- Cell lines --- p.84 / Chapter 5.2.2 --- Normal and primary tumor tissues --- p.85 / Chapter 5.3 --- Results and Discussion --- p.86 / Chapter 5.3.1 --- Expression profiling of TUSC14 in normal tissues and tumor cell lines --- p.86 / Chapter 5.3.2 --- Frequent inactivation of TUSC14 by promoter CpG methylation --- p.90 / Chapter 5.3.3 --- Pharmacologic and genetic demethylation restores TUSC14 expression --- p.94 / Chapter 5.3.4 --- Frequent TUSC14 methylation in primary tumors --- p.96 / Chapter 5.4 --- Summary --- p.98 / Chapter CHAPTER 6 --- Characterization of the Tumor Suppressive Functions of TUSC14 --- p.99 / Chapter 6.1 --- Introduction --- p.91 / Chapter 6.2 --- Materials and Methods --- p.100 / Chapter 6.2.1 --- Gene cloning and plasmids construction of TUSC14 --- p.100 / Chapter 6.2.2 --- Drug and stress treatments of cells --- p.100 / Chapter 6.3 --- Results and Discussion --- p.102 / Chapter 6.3.1 --- TUSC14 localizes to nuclear speckles --- p.102 / Chapter 6.3.2 --- TUSC14 inhibits clonogenicity --- p.107 / Chapter 6.3.3 --- Expression of TUSC14 induces apoptosis in tumor cells --- p.110 / Chapter 6.3.4 --- TUSC14 alters cell cycle progression --- p.112 / Chapter 6.3.5 --- TUSC14 acts as a transcriptional repressor of multiple genes --- p.114 / Chapter 6.3.6 --- TUSC14 represses NF-кB activity through an HDAC-dependent mechanism --- p.118 / Chapter 6.3.7 --- The effect of TUSC14 on the expression of downstream targets of NF-κB Signaling --- p.120 / Chapter 6.3.8 --- TUSC14 co-immunoprecipitates with HDAC1 and p65 --- p.124 / Chapter 6.3.9 --- ChIP analysis of promoters of TUSC14-regulated genes --- p.127 / Chapter 6.4 --- Summary --- p.130 / Chapter CHAPTER 7 --- General Discussion --- p.133 / Chapter CHAPTER 8 --- Conclusions --- p.138 / References --- p.140

Zheng Ping-pin. / Thesis submitted in: December 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 118-159). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT(ENGLISH/CHINESE) --- p.iii / CONTENTS --- p.viii / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xii / PUBLICATION --- p.xiii / Chapter CHAPTER I --- INTRODUCTION / Chapter I.1. --- Preface --- p.1 / Chapter I.2. --- Overview of Carcinogenesis --- p.2 / Chapter I.3. --- Oncogene --- p.5 / Chapter I.4. --- Tumor Suppressor Genes (TSGs) --- p.6 / Chapter I.5 --- Detection of Oncogene and Tumor Suppressor Genes --- p.9 / Chapter I.5.1 --- Detaction of Oncogene --- p.9 / Chapter I.5.2. --- Detection of Tumor Suppressor Genes --- p.11 / Chapter I.6. --- Profiles of Oncogenes/TSGs and Molecular Subtype about Astrocytic Tumors --- p.17 / Chapter I.7. --- Intratumoral Heterogeneity and Microsatellite Instability --- p.20 / Chapter I.8. --- Outline of Ependymoma --- p.20 / Chapter I.9. --- Clinicopathological Factors and Prognosis --- p.22 / Chapter I.9.1. --- Histology and Grading (2000) --- p.22 / Chapter I.9.2. --- Prognosis Factors --- p.23 / Chapter I.9.2.1. --- Age/Sex/Location --- p.23 / Chapter I.9.2.2. --- Extent of Resection --- p.25 / Chapter I.9.2.3. --- Radiotherapy and Chemotherapy --- p.25 / Chapter I.9.2.4. --- Histology --- p.26 / Chapter I.10. --- "Cytogenetic, Molecular Genetic and Molecular Studies" --- p.27 / Chapter I.11. --- Advantages and Disadvantages of The Research Methods --- p.34 / Chapter CHAPTER II --- AIM OF STUDY --- p.36 / Chapter CHAPTER III --- MATERIALS AND METHODS --- p.37 / Chapter III.1. --- Tumor Samples and DNA Preparations --- p.37 / Chapter III.1.1. --- Tumor Samples --- p.38 / Chapter III.1.2. --- DNA Preparation --- p.38 / Chapter III.2. --- Comparative Genomic Hybridization --- p.42 / Chapter III.2.1. --- Metaphase Preparation --- p.42 / Chapter III.2.2. --- "DNA Labeling, Hybridization, and Detection" --- p.43 / Chapter III.2.3. --- Digital Image Analysis --- p.45 / Chapter III.3 --- High-Resolution Allelotying (Microsatellite Analysis) --- p.46 / Chapter III.3.1 --- General Outline --- p.46 / Chapter III.3.2 --- Multiplex PCR --- p.47 / Chapter III.3.3 --- Pooling of PCR Products --- p.49 / Chapter III.3.4 --- Electrophoresis --- p.50 / Chapter III.3.5. --- Assessment of Allelic Imbalance by Calculating Allelic Ratio --- p.52 / Chapter III.3.6 --- Standards of Evalution --- p.53 / Chapter III.3.7 --- Separating Allelic Loss from Allelic Duplication --- p.54 / Chapter III.3.8 --- Statistical Analyses --- p.54 / Chapter CHAPTER IV --- RESULTS --- p.54 / Chapter IV.1. --- CGH Study --- p.54 / Chapter IV.1.1 --- Overview --- p.54 / Chapter IV.1.2 --- Common Deletion Regions --- p.58 / Chapter IV.1.3 --- Common duplication Regions --- p.60 / Chapter IV.2. --- High-Resolution Allelotyping (Microsatellite Analysis) --- p.60 / Chapter IV.2.1. --- Overview of Results --- p.60 / Chapter IV.2.2. --- LOH profile of Individual Chromosome --- p.93 / Chapter IV.2.3. --- Overlapping Small Deletion Regions --- p.95 / Chapter CHAPTER V --- DISCUSSION --- p.97 / Chapter V.1. --- . General Outline --- p.98 / Chapter V.2. --- Chromosome 22 --- p.99 / Chapter V.3. --- Chromosome 17 --- p.102 / Chapter V.4. --- Chromosome 6 --- p.104 / Chapter V.5. --- Chromosome 16 --- p.105 / Chapter V.6. --- Chromosome 19 --- p.107 / Chapter V.7. --- Chromosome 20 --- p.108 / Chapter V.8. --- Chromosome 7 --- p.109 / Chapter V.9. --- Chromosome 12 --- p.110 / Chapter V.10. --- Chromosome 9 --- p.111 / Chapter V.11. --- Chromosome 5 --- p.112 / Chapter V.12. --- Chromosome 4 --- p.112 / Chapter V.13. --- Correlation of CGH with Allelotyping in the Study --- p.112 / Chapter V.14. --- Conclusion --- p.114 / Chapter CHAPTER VI --- LIMITATIONS OF THE STUDY --- p.115 / Chapter CHAPTER VII --- FUTURE STUDY --- p.116 / REFERENCES --- p.118

Submitted by NAZEL OLIVEIRA FILHO null (nazelfilho@gmail.com) on 2017-03-05T19:28:40Z No. of bitstreams: 1 Nazel Oliveira Filho (Doutorado)1.pdf: 1148840 bytes, checksum: dafa36f0e3c6248373d61e2c7a5b572b (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-09T14:30:20Z (GMT) No. of bitstreams: 1 oliveirafilho_n_dr_bot.pdf: 1148840 bytes, checksum: dafa36f0e3c6248373d61e2c7a5b572b (MD5) / Made available in DSpace on 2017-03-09T14:30:20Z (GMT). No. of bitstreams: 1 oliveirafilho_n_dr_bot.pdf: 1148840 bytes, checksum: dafa36f0e3c6248373d61e2c7a5b572b (MD5) Previous issue date: 2017-02-10 / INTRODUÇÃO A maioria dos estudos que avaliou a influência da hiperglicemia perioperatória na ocorrência de infecção pós-operatória em pacientes submetidos a neurocirurgias foram realizados em pacientes graves e estudaram apenas infecção de ferida cirúrgica. A hipótese deste estudo é que pacientes submetidos à craniotomia eletiva para ressecção de tumores cerebrais que apresentarem hiperglicemia no perioperatório terão um maior risco de evoluir com complicações infecciosas no pós-operatório. OBJETIVOS Avaliar a associação entre hiperglicemia perioperatória (glicemia ≥ 180 mg/dl) e a ocorrência de complicações infecciosas no pós-operatório. Objetivos secundários: avaliar a associação entre hiperglicemia pós-operatória e tempo de internamento na UTI e no hospital e a influência do tipo de anestesia (geral balanceada X anestesia venosa total) nos níveis glicêmicos perioperatórios. METODOLOGIA Coorte retrospectiva envolvendo todos pacientes adultos submetidos à craniotomia eletiva para ressecção de tumores cerebrais entre janeiro de 2009 e dezembro de 2015 no Hospital São Rafael, um hospital terciário em Salvador, Bahia. Dados demográficos e informações do intraoperatório e pós-operatório foram coletados de prontuários através de ficha padronizada. Foram avaliados os valores de glicemia no pré-operatório, intraoperatório e no 1º dia de pós-operatório. Regressão logística binária foi realizada para identificar os fatores de risco independentes para a ocorrência de infecção pós-operatória. Efeitos e diferenças foram considerados estatisticamente significativos se p < 0,05. RESULTADOS Foram incluídos no estudo 505 pacientes. Cinquenta e sete (11,3%) apresentaram complicação infecciosa no pós-operatório. Os pacientes que evoluíram com infecção no pós-operatório tiveram um maior tempo de internamento na UTI e no hospital e uma maior taxa de óbito no pós-operatório. Análise univariável demonstrou que comorbidade, uso pré-operatório de corticosteroides, uso de drogas vasoativas no intraoperatório, transfusão sanguínea intraoperatória, duração cirúrgica > 210 minutos, implante para derivação ventricular externa, glicemia admissão na UTI ≥ 180mg/dl e pico glicêmico ≥ 180mg/dL no 1º DPO foram associadas à infecção pós-operatória. Após análise multivariável, os níveis glicêmicos perioperatórios ≥ 180mg/dL não foram confirmados como fatores de risco independentes para a ocorrência de complicação infecciosa no pós-operatório. Os pacientes submetidos à anestesia venosa total apresentaram menores níveis glicêmicos no intraoperatório e no momento da admissão na UTI do que os que foram submetidos à anestesia geral balanceada. Tempo de internamento na UTI e no hospital foram semelhantes entre os pacientes que evoluíram ou não com pico glicêmico ≥ 180mg/dL no 1º DPO. CONCLUSÃO Pacientes adultos submetidos à craniotomia eletiva para ressecção de tumores cerebrais que apresentaram complicações infecciosas no pós-operatório tiveram maiores níveis glicêmicos perioperatórios, apresentaram um maior tempo de internação na UTI e no hospital e uma maior taxa de óbito. A anestesia venosa total determinou redução dos valores de glicemias no intraoperatório e no momento admissão na UTI. Após análise multivariável os níveis glicêmicos perioperatórios ≥ 180mg/dL não estiveram associados a um maior risco de infecção pós-operatória. Estudos prospectivos e multicêntricos são necessários para confirmar nossos resultados.

Os tumores odontogênicos são um grupo heterogéneo de lesões formadas a partir de tecidos que dão origem ao dente. A metilação do ADN, uma adição covalente de um grupo metilo na posição 5 de carbono de um nucleótideo de citosina, é considerado um importante regulador da expressão génica. A adição do radical metil é catalisada por ADN metiltransferases (DNMTs). Embora alguns estudos epigenéticos tenham sido realizados em tumores odontogênicos, um estudo com os três tipos de DNMTs em vários membros desse grupo está em falta. Este estudo analisa a expressão de DNMTs em tumores odontogênicos. Amostras de vinte ameloblastomas, dez Calcificante tumores odontogênicos císticos, dez calcificados tumores epiteliais, dez tumor odontogênico adenomatóide, dez tumores odontogênicos queratocísticos, quatro fibromas ameloblásticos, dois fibro-odontoma ameloblástico, quatro fibroma centrais odontogênicos, sete tecidos de fibromas odontogênicos periféricos e dez mixomas odontogênicos foram incluídos. DNMT1, 3A e 3B foram expressas no núcleo e / ou citoplasma de todos os tumores odontogênicos. A alta expressão de DNMTs em células de tumor odontogênico sugere metilação como um mecanismo importante para este grupo de tumores. / Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalyzed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyzes the expression of DNMTs in odontogenic tumours. Formalin-fixed and paraffin-embedded tissue samples of twenty ameloblastomas, ten calcifying cystic odontogenic tumors, ten calcifying epithelial tumors, ten adenomatoid odontogenic tumors, ten keratocystic odontogenic tumors, five ameloblastic fibromas, two ameloblastic fibro-odontoma, four central odontogenic fibroma, seven peripheral odontogenic fibroma and ten odontogenic mixoma were included. DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.

Lee, Kwan Yeung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-113). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Content --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / List of papers published during the study --- p.xvi / Chapter Chapter 1 --- Introduction and Aim of Study --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Project objective and potential significances --- p.5 / Chapter Chapter 2 --- Literatures Review --- p.6 / Chapter 2.1 --- Cancer genetics and Tumor suppressor genes --- p.6 / Chapter 2.2 --- Epigenetic --- p.7 / Chapter 2.2.1 --- DNA methylation and promoter CpG island --- p.8 / Chapter 2.2.2 --- Establishment and maintenance of DNA methylation --- p.9 / Chapter 2.2.3 --- Transcriptional silencing by DNA hypermethylation --- p.9 / Chapter 2.3 --- Cancer epigenetic --- p.11 / Chapter 2.3.1 --- Hypomethylation of the cancer genome --- p.12 / Chapter 2.3.2 --- Hypermethylation in cancers --- p.12 / Chapter 2.3.3 --- Clinical relevance of cancer epigenetic --- p.13 / Chapter 2.4 --- Nasopharyngeal carcinoma --- p.14 / Chapter 2.4.1 --- NPC genetic and epigenetic --- p.15 / Chapter 2.5 --- 12p as a putative tumor suppressor locus --- p.16 / Chapter 2.5.1 --- Hematological malignancies associated with 12p loss --- p.17 / Chapter 2.5.2 --- Prostate cancer associated with 12p loss --- p.20 / Chapter 2.5.3 --- Lung cancer associated with 12p loss --- p.22 / Chapter 2.5.4 --- 12p deletion in other cancers --- p.23 / Chapter 2.6 --- 16q as a tumor suppressor locus --- p.24 / Chapter 2.6.1 --- Breast cancer and 16q --- p.25 / Chapter 2.6.2 --- Loss of 16q and prostate cancer --- p.26 / Chapter 2.6.3 --- Loss of 16q and hepatocellular carcinoma --- p.28 / Chapter 2.6.4 --- 16q deletion associated with other cancers --- p.29 / Chapter Chapter 3 --- Materials and Methods --- p.30 / Chapter 3.1 --- Cell lines and tissue samples --- p.30 / Chapter 3.1.1 --- Cell lines --- p.30 / Chapter 3.1.2 --- Maintenance of cell lines --- p.31 / Chapter 3.1.3 --- Drugs treatment of cell lines --- p.31 / Chapter 3.1.4 --- Normal tissues --- p.32 / Chapter 3.1.5 --- Total RNA extraction --- p.32 / Chapter 3.1.6 --- Genomic DNA extraction --- p.32 / Chapter 3.2 --- General techniques --- p.33 / Chapter 3.2.2 --- TA cloning and blunt end cloning of PCR product --- p.33 / Chapter 3.2.3 --- Transformation of cloning products to E. coli competent cells --- p.34 / Chapter 3.2.4 --- Preparation of plasmid DNA --- p.34 / Chapter 3.2.4.1 --- Mini-prep plasmid DNA extraction --- p.34 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.35 / Chapter 3.2.5 --- Measurement of DNA or RNA concentrations --- p.36 / Chapter 3.2.6 --- DNA sequencing of plasmid DNA and PCR products --- p.36 / Chapter 3.3 --- Preparation of reagents and medium --- p.37 / Chapter 3.4 --- Semi-quantitative Reverse-Transcription (RT) PCR expression analysis --- p.38 / Chapter 3.4.1 --- Reverse transcription reaction --- p.38 / Chapter 3.4.2 --- Semi-quantitative RT-PCR --- p.39 / Chapter 3.4.2.1 --- Primers design --- p.39 / Chapter 3.4.2.2 --- PCR reaction --- p.39 / Chapter 3.5 --- Methylation analysis of candidate genes --- p.40 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.41 / Chapter 3.5.2 --- Methylation-specific PCR (MSP) --- p.42 / Chapter 3.5.2.1 --- Bioinformatics prediction of CpG island --- p.42 / Chapter 3.5.2.2 --- Primers design --- p.42 / Chapter 3.5.2.3 --- PCR reaction --- p.42 / Chapter 3.5.3 --- Bisulfite Genomic Sequencing (BGS) --- p.43 / Chapter 3.5.3.1 --- Primers design --- p.43 / Chapter 3.5.3.2 --- PCR reaction --- p.44 / Chapter 3.6 --- Construction of expression vectors of candidate genes --- p.44 / Chapter 3.6.1 --- Construction of IRF8 expression vector --- p.44 / Chapter 3.6.2 --- Construction of PTPRO expression vector --- p.44 / Chapter 3.6.2.1 --- Experimental design --- p.44 / Chapter 3.6.2.2 --- PCR and cloning of PCR products --- p.46 / Chapter 3.6.2.3 --- Restriction digestion of cloning vectors and expression vector --- p.48 / Chapter 3.6.2.4 --- Ligation of cloning fragments --- p.48 / Chapter 3.7 --- Colony formation assay on monolayer culture --- p.48 / Chapter 3.8 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Identification of candidate TSGs in deleted regions --- p.50 / Chapter 4.1 --- Research plan --- p.50 / Chapter 4.2 --- Results --- p.50 / Chapter 4.2.1 --- Mapping of the deleted B AC clones on their chromosomal locations --- p.50 / Chapter 4.2.2 --- Identification of down-regulated genes in NPC by semi-quantitative RT-PCR analysis --- p.51 / Chapter 4.3 --- Discussion --- p.55 / Chapter Chapter 5 --- Tumor suppressor function studies of candidate TSGs --- p.60 / Chapter 5.1 --- Research plan --- p.60 / Chapter 5.2. --- IRF8 is the 16q candidate TSG --- p.60 / Chapter 5.2.1 --- Frequent silencing of IRF8 mRNA expression in multiple carcinomas --- p.60 / Chapter 5.2.2 --- Methylation status of IRF8 promoter region correlated with its transcriptional silencing --- p.62 / Chapter 5.2.3 --- Restoration of IRF8 expression by pharmacological and genetic demethylation --- p.65 / Chapter 5.2.4 --- IRF8 inhibited the anchorage dependent growth of tumor cells on monolayer culture --- p.67 / Chapter 5.2.5 --- Discussion --- p.68 / Chapter 5.3 --- PTPRO is the down-regulated target at 12pl3.2-12.3 tumor suppressor locus --- p.73 / Chapter 5.3.1 --- Frequent silencing of PTPRO in multiple carcinoma cell lines --- p.73 / Chapter 5.3.2 --- Frequent methylation of PTPRO promoter CpG island in multiple carcinoma cell lines correlated with its reduced expression --- p.74 / Chapter 5.3.3 --- Re-expression of PTPRO by pharmacological and genetic demethylation --- p.77 / Chapter 5.3.4 --- PTPRO inhibited the growth of tumor cells in vitro --- p.79 / Chapter 5.3.5 --- Discussion --- p.81 / Chapter 5.4 --- RERG is another candidate TSG in 12pl3.2 - 12.3 region --- p.87 / Chapter 5.4.1 --- Down-regulation of RERG mRNA expression in carcinoma cell line --- p.87 / Chapter 5.4.2 --- Hypermethylation of RERG promoter is a frequent event in multiple carcinomas --- p.88 / Chapter 5.4.3 --- Re-expression of RERG mRNA following pharmacological and genetic demethylation --- p.90 / Chapter 5.4.4 --- Discussion --- p.92 / Chapter Chapter 6 --- General discussion --- p.96 / Chapter Chapter 7 --- Summary --- p.101 / Reference --- p.103

Ng, Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-113). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of abbreviations --- p.ii / List of Tables --- p.iv / List of Figures --- p.v / List of Publications --- p.viii / Abstract in English --- p.ix / Abstract in Chinese --- p.xi / Table of Contents --- p.xiii / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Tumor suppressor genes (TSGs) and the modes of TSG inactivation during carcinogenesis --- p.1 / Chapter 1.2 --- Epigenetic modifications --- p.3 / Chapter 1.2.1 --- DNA methylation --- p.4 / Chapter 1.2.1a --- Establishment of DNA methylation patterns and DNA methyltransferases --- p.5 / Chapter 1.2.1b --- DNA hypermethylation and carcinogenesis --- p.6 / Chapter 1.2.1c --- Mechanism for gene silencing by CpG methylation --- p.6 / Chapter 1.2.1d --- DNA hypomethylation and carcinogenesis --- p.10 / Chapter 1.2.1e --- Loss of imprinting and carcinogenesis --- p.11 / Chapter 1.2.1f --- Potential factors leading to aberrant methylation patterns in cancers --- p.12 / Chapter 1.2.2 --- Deregulation of histone modifications and carcinogenesis --- p.14 / Chapter 1.2.3 --- Interplay between chromatin modifications and DNA methylation --- p.15 / Chapter 1.3 --- Identification of tumor suppressor genes (TSGs) --- p.17 / Chapter 1.4 --- Nasopharyngeal carcinoma as a cancer model of the current project --- p.18 / Chapter 1.5 --- Genetic and epigenetic changes in NPC --- p.19 / Chapter 1.6 --- Involvement of 5qll-ql2 and 14q32 in carcinogenesis --- p.22 / Chapter 1.6.1 --- Chromosome 5ql l-ql2 and carcinogenesis --- p.22 / Chapter 1.6.2 --- Chromosome 14q32 and carcinogenesis --- p.24 / Chapter 1.7 --- Clinical implications of epigenetics in cancers --- p.27 / Chapter Chapter 2 --- Aims of study and Research plan --- p.31 / Chapter Chapter 3 --- Materials and Methods --- p.34 / Chapter 3.1 --- Cell lines and Normal Tissues --- p.35 / Chapter 3.2 --- Routine cell line maintenance --- p.35 / Chapter 3.3 --- Drug treatments --- p.35 / Chapter 3.4 --- Total RNA extraction --- p.35 / Chapter 3.5 --- Genomic DNA extraction --- p.36 / Chapter 3.6 --- General techniques --- p.37 / Chapter 3.6.1 --- Gel electrophoresis --- p.37 / Chapter 3.6.2 --- DNA and RNA quantification --- p.37 / Chapter 3.6.3 --- LB medium and LB plate preparation --- p.38 / Chapter 3.6.4 --- Plasmid extraction --- p.38 / Chapter 3.6.4a --- Mini-scale preparation of plasmid DNA --- p.38 / Chapter 3.6.4b --- Large-scale preparation of endotoxin-free plasmid DNA --- p.39 / Chapter 3.6.5 --- DNA sequencing --- p.39 / Chapter 3.7 --- Reverse transcription-PCR (RT-PCR) --- p.40 / Chapter 3.7.1 --- Reverse transcription (RT) --- p.40 / Chapter 3.7.2 --- Semi-quantitative RT-PCR --- p.41 / Chapter 3.8 --- Methylation analysis --- p.42 / Chapter 3.8.1 --- Sodium bisulfite modification of DNA --- p.42 / Chapter 3.8.2 --- CpG island analysis --- p.42 / Chapter 3.8.3 --- Methylation-specific PCR (MSP) --- p.43 / Chapter 3.8.4 --- Bisulfite genomic sequencing (BGS) --- p.44 / Chapter 3.9 --- Construction of expression plasmids --- p.45 / Chapter 3.9.1 --- Construction of the MGC80-expressing vector --- p.45 / Chapter 3.9.2 --- Construction of the TUSC14-expressing vector --- p.46 / Chapter 3.10 --- Functional analyses --- p.47 / Chapter 3.10.1 --- Monolayer colony formation assay --- p.47 / Chapter 3.10.2 --- Soft agar assay --- p.48 / Chapter 3.11 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Results --- p.50 / Chapter 4.1 --- Identification of 5qll-ql2 and 14q32.2-q32.32 as frequently deleted regions in NPC by aCGH --- p.50 / Chapter 4.2 --- Identification of novel candidate TSGs at chromosome 5qll-ql2 through integrative genomics and epigenetics --- p.51 / Chapter 4.2.1 --- Expression profiling of the candidate genes at 5ql l-ql2 in NPC cell lines --- p.51 / Chapter 4.2.2 --- MGC80 as a target of study at 5ql2 --- p.54 / Chapter 4.2.2a --- Ubiquitous expression in normal human tissues and frequent down-regulation of MGC80 in multiple tumor cell lines --- p.54 / Chapter 4.2.2b --- Methylation analysis of MGC80 --- p.56 / Chapter 4.2.2c --- Restoration of MGC80 expression after pharmacologic and genetic demethylation --- p.59 / Chapter 4.2.2d --- Functional study of MGC80 in multiple carcinomas --- p.61 / Chapter 4.2.2e --- Discussion --- p.63 / Chapter 4.2.3 --- TUSC14 as a target of study at 5ql2 --- p.67 / Chapter 4.2.3a --- TUSC14 was broadly expressed in normal human tissues and frequently down-regulated in multiple tumor cell lines --- p.67 / Chapter 4.2.3b --- Methylation analysis of TUSCI4 --- p.69 / Chapter 4.2.3c --- Pharmacologic and genetic demethylation reactivated TUSC14 expression --- p.72 / Chapter 4.2.3d --- Functional study ofTUSC14 in multiple carcinomas --- p.74 / Chapter 4.2.3e --- Discussion --- p.76 / Chapter 4.3 --- Identification of candidate TSGs at chromosome 14q32 through integrative genomics and epigenetics --- p.80 / Chapter 4.3.1 --- Expression profiling of the candidate genes at 14q32 in NPC cell lines --- p.80 / Chapter 4.3.2 --- DLK1 as a target of study at 14q32 --- p.82 / Chapter 4.3.2a --- Expression analysis of DLK1 in normal tissues and NPC cell lines --- p.82 / Chapter 4.3.2b --- Methylation analysis ofDLKl in NPC --- p.83 / Chapter 4.3.2c --- Restoration of DLK1 expression after pharmacologic demethylation --- p.84 / Chapter 4.3.2d --- Functional study ofDLKl in NPC --- p.85 / Chapter 4.3.2e --- Discussion --- p.87 / Chapter Chapter 5 --- General discussion --- p.92 / Chapter Chapter 6 --- Summary --- p.99 / Chapter Chapter 7 --- Future study --- p.101 / Reference list --- p.103

Chondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. To achieve a greater understanding of chondrosarcoma tumorigenesis, a model for human chondrosarcoma has been established in a rat system. The model, known as the Swarm rat chondrosarcoma (SRC), resembles human chondrosarcoma and provides a system to study tumor growth and progression. Here we examined the influence of the tumor microenvironment and the impact of genome-wide hypomethylation on the behavior of SRC tumors, two factors known to contribute fundamentally to the development and progression of solid tumors. Previous studies with SRC revealed that tumor microenvironment can significantly influence chondrosarcoma malignancy, but the underlying biologic mechanisms have not been defined. To address this issue we carried out epigenetic and gene expression studies on the SRC tumors that were initiated at different transplantation sites. The epigenetic analysis revealed that microenvironmental changes could promote global DNA hypomethylation in SRC cells. Subsequent gene expression analyses revealed that the transplantation site had a significant impact on the gene expression profiles of SRC tumors. These SRC tumors had unique gene expression profiles, and we were able to identify genes that were differentially expressed between SRC tumors originating from different transplantation sites. Functional analyses of two differentially expressed genes, thymosin-beta-4 and c-fos, provided insight into the role that these genes may play in the development and progression of chondrosarcoma.

The retinoblastoma tumor suppressor protein, Rb, is a key regulator of the mammalian cell cycle and its inactivation facilitates S-phase entry. Rb is inactivated through multiple waves of phosphorylation, mediated mainly by kinases associated with D and E type cyclins in the G1 phase of the cell cycle. Our earlier studies had shown that the signaling kinase Raf-1 (c-Raf) physically interacts with Rb upon growth factor stimulation and initiates the phosphorylation cascade. We had shown that an 8 amino acid peptide derived from Raf-1 could disrupt the Rb-Raf-1 interaction leading to an inhibition of Rb phosphorylation, cell proliferation and tumor growth in nude mice. Here, we describe a newly identified orally-active small molecule, RRD-251 (Rb - Raf-1 Disruptor 251), that disrupts potently and selectively the binding of Raf-1 to Rb; it had no effect on Rb-HDAC1, Rb-Prohibitin, Rb-Ask1, Rb-cyclin E, or Raf-1-Mek interactions. RRD-251 inhibited anchorage-dependent and -independent growth of human cancer cells; it could also potently inhibit angiogenesis both in vitro and in vivo. Oral or intra-peritoneal administration of RRD-251 resulted in a significant suppression of growth of tumors xenotransplanted into athymic nude mice; the tumor suppressive effects were restricted to tumors carrying a wild-type Rb gene. Thus, selective targeting of Rb-Raf-1 interaction appears to be a promising approach for developing novel anti-cancer agents. In addition to mitogens, tobacco components like NNK and nicotine can induce cell proliferation and angiogenesis, contributing to lung cancer. Induction of cell proliferation by tobacco components required the binding of Raf-1 to Rb and RRD-251 could prevent nicotine induced cell proliferation. Our studies also show how nicotine not only promotes tumor growth in vivo, it also increases chance of tumor recurrence and metastasis. In addition to growth factors and tobacco components, cytokines like TNFα could induce Rb-Raf-1 interaction in vascular smooth muscle cells. Since TNFα-induced proliferation of vascular smooth muscle cells contributes to growth of atherosclerotic plaques, RRD-251 could be beneficial in controlling atherosclerosis as well. Thus, it appears that drugs that can disrupt the Rb-Raf-1 interaction might have beneficial effects in a wide spectrum of human diseases.