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Growth gone awry: exploring the role of embryonic liver development genes in HCV induced cirrhosis and hepatocellular carcinomaBehnke, Martha K. 19 November 2012 (has links)
Introduction and methods: Hepatocellular carcinoma (HCC) remains a difficult disease to study even after a decade of genomic analysis. Metabolic and cell-cycle perturbations are known, large changes in tumors that add little to our understanding of the development of tumors, but generate “noise” that obscures potentially important smaller scale expression changes in “driver genes”. Recently, some researchers have suggested that HCC shares pathways involving the master regulators of embryonic development. Here, we investigated the involvement and specificity of developmental genes in HCV-cirrhosis and HCV-HCC. We obtained microarray studies from 30 patients with HCV-cirrhosis and 49 patients with HCV-HCC and compared to 12 normal livers. Differential gene expression is specific to liver development genes: 86 of 202 (43%) genes specific to liver development had differential expression between normal and cirrhotic or HCC samples. Of 60 genes with paralogous function, which are specific to development of other organs and have known associations with other cancer types, none were expressed in either adult normal liver or tumor tissue. Developmental genes are widely differentially expressed in both cirrhosis and early HCC, but not late HCC: 69 liver development genes were differentially expressed in cirrhosis, and 58 of these (84%) were also dysregulated in early HCC. 19/58 (33%) had larger-magnitude changes in cirrhosis and 5 (9%) had larger-magnitude changes in early HCC. 16 (9%) genes were uniquely altered in early tumors, while only 2 genes were uniquely changed in late-stage (T3 and T4) HCC. Together, these results suggest that the involvement of the master regulators of liver development are active in the pre-cancerous cirrhotic liver and in cirrhotic livers with emerging tumors but play a limited role in the transition from early to late stage HCC. Common patterns of coordinated developmental gene expression include: (1) Dysregulation of BMP2 signaling in cirrhosis followed by overexpression of BMP inhibitors in HCC. BMP inhibitor GPC3 was overexpressed in nearly all tumors, while GREM1 was associated specifically with recurrence-free survival after ablation and transplant. (2) Cirrhosis tissues acquire a progenitor-like signature including high expression of Vimentin, EPCAM, and KRT19, and these markers remain over-expressed to a lesser extent in HCC. (3) Hepatocyte proliferation inhibitors (HPI) E-cadherin (CDH1), BMP2, and MST1 were highly expressed in cirrhosis and remained over-expressed in 16 HCC patients who were transplanted with excellent recurrence-free survival (94% survival after 2 years; mean recurrence-free survival = 5.6 yrs), while loss in early HCC was associated with early recurrence and (2 year). Loss of HPI overexpression was also correlated with overexpression of c-MET and loss of STAT3, LAMA2, FGFR2, CITED2, KIT, SMAD7, GATA6, ERBB2, and NOTCH2.
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Rôle de la reptine dans le carcinome hépatocellulaire / Role of Reptin in hepatocellular carcinomaGrigoletto, Aude 30 November 2012 (has links)
Le carcinome hépatocellulaire (CHC) est le principal cancer primitif du foie et est associé à un très mauvais pronostic. Notre équipe a mis en évidence que la Reptine et la Pontine, des AAA+ ATPases homologues, sont surexprimées dans le CHC par rapport au foie non tumoral. Au cours de ce travail de thèse, j’ai contribué à démontrer que l’extinction de la Reptine par l’induction de shRNA suffit à arrêter la croissance de tumeurs déjà établies, et même à induire leur régression dans des xénogreffes chez la souris. Ces résultats encourageants suggèrent que la Reptine pourrait être une cible thérapeutique dans le CHC. L’utilisation de siRNA en thérapeutique n’étant pas envisageable actuellement, il parait plus pertinent de tenter de cibler la Reptine via son activité ATPase. Le principal objectif de ma thèse était donc de déterminer l’implication de l’activité ATPase de la Reptine pour ses propriétés oncogéniques dans le CHC. Nos résultats ont montré que des mutants inactifs de la Reptine (D299N et E300G) ont un effet dominant négatif et ne sont pas capables de complémenter l’absence de la Reptine endogène, ce qui conduit à une diminution significative de la croissance des cellules HuH7 et Hep3B, et à une induction de l’apoptose. Ceci indique que l’activité ATPase de la Reptine est nécessaire pour la croissance et la survie des cellules de CHC. Enfin, grâce à une étude transcriptomique, nous avons identifié de nouveaux gènes dont l’expression est régulée par la Reptine et/ou la Pontine. Parmi ces gènes, certains pourraient être impliqués dans les fonctions oncogéniques de la Reptine et/ou de la Pontine dans le CHC. Finalement, ce travail a permis de mettre en évidence l’implication de l’activité ATPase de la Reptine, et d’apporter des éléments permettant de mieux comprendre le mécanisme d’action de la Reptine dans le CHC. / Hepatocellular carcinoma (HCC) is the main primary cancer of the liver and is often associated with poor prognosis. Our team has demonstrated that Reptin and Pontin, two AA+ ATPases, are overexpressed in HCC compared to normal liver. Moreover this overexpression is also associated with poor prognosis. In the course of my PhD, I demonstrated that shRNA-mediated silencing of Reptin is sufficient to inhibit tumor growth and even can promote their regression in xenografted mice. These encouraging results suggest that Reptin might represent a novel therapeutic target in HCC. As the use of siRNA as therapeutic tools is still debated, the targeting of Reptin enzymatic activity might represent a more relevant approach to impair its functions. To this end I first proposed to determine the involvement of Reptin ATPase activity in HCC oncogenesis. My results show that ATPase inactive Reptin mutants (D299N and E300G) play dominant negative roles toward Reptin functions and are unable to complement for the depletion of endogenous Reptin, thereby leading to a significant decrease of cell growth and to a significant increase of apoptosis in HuH7 and Hep3B cells. These results show that Reptin’s ATPase activity is necessary for HCC cell growth and survival. Moreover, using a transcriptomic approach that compared gene expression upon siRNA-mediated Reptin or Pontin silencing, we identified specific genes whose expression is under the control of those proteins and whose functions might provide mechanistic explanation to Reptin’s involvement in HCC. Collectively, the results obtained during my PhD thesis have characterized the contribution of Reptin ATPase activity to HCC growth and development and might represent a founding step in the understanding of Reptin’s biology in cancer development.
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Análise da sobrevida de pacientes com carcinoma hepatocelular pequeno / Survival analysis of patients with small hepatocellular carcinomaKikuchi, Luciana Oba Onishi 21 November 2007 (has links)
Introdução: O carcinoma hepatocelular (CHC) é o câncer primário de fígado mais comum. A cirrose hepática é o principal fator de risco para esse tumor. O rastreamento para o CHC em pacientes com cirrose tem sido recomendado há anos. Acredita-se que a detecção e o tratamento precoce do CHC melhorem a sobrevida dos pacientes. O objetivo deste estudo foi analisar a sobrevida dos pacientes cirróticos com CHC pequeno e identificar fatores preditivos de sobrevida no Brasil. Casuística e Métodos: Entre janeiro de 1998 e dezembro de 2003, 74 pacientes cirróticos com CHC foram avaliados. Eles preenchiam os seguintes critérios: CHC com até três nódulos e no máximo 30 mm de diâmetro cada. Os fatores preditores de sobrevida foram identificados através do método de Kaplan-Meier e o modelo de Cox. Resultados: A média de idade foi de 58 anos (32-77); 71% dos pacientes eram do sexo masculino; 64% tinham hepatite C; 60% eram Child-Pugh A, o valor mediano da pontuação de MELD foi de 11; 79% tinham hipertensão portal. No momento do diagnóstico, 71% tinham uma única lesão; o tamanho do principal tumor era menor que 20 mm em 47%; o valor médio de AFP foi de 131 ng/ml. Três pacientes tinham trombose de veia porta, sugestiva de invasão vascular. Cinqüenta pacientes (67,5%) foram incluídos na lista de transplante hepático, que foi realizado só em quatro pacientes. A ressecção cirúrgica do tumor foi possível em quatro pacientes. Quarenta e oito (64,8%) pacientes receberam tratamento ablativo percutâneo (ablação por radiofreqüência ou injeção percutânea de etanol). Nove pacientes não receberam nenhum tratamento específico para o tumor. A taxa de sobrevida geral foi de 80%; 62%; 41% e 17% em 12, 24, 36 e sessenta meses, respectivamente. O tempo médio de seguimento após o diagnóstico do CHC foi de 23 meses (mediana de 22 meses, variando de um a 86 meses) para todo o grupo. Durante o seguimento, ocorreram 39 óbitos ocorreram relacionados com insuficiência hepática ou progressão do CHC. A análise univariada dos 74 pacientes mostrou que escore MELD maior que 11 (p = 0,016), classificação de Child-Pugh B e C (p = 0,007), AFP > 100 ng/ml (p = 0,006), mais de uma lesão (p = 0,041), diâmetro do tumor > 20 mm (p = 0,009) e presença de invasão vascular (p < 0,0001) foram preditores independentes de sobrevida. A análise de regressão de Cox identificou invasão vascular (RR = 14,60 - IC 95% = 3,3 - 64,56 - p < 0,001) e tamanho do tumor > 20mm (RR = 2,14 - IC 95% = 1,07 - 4,2 - p = 0,030) como preditores independentes de pior sobrevida. O tratamento do CHC esteve relacionado com melhor sobrevida. Conclusão: A identificação de CHC pequeno com até 20 mm de diâmetro está relacionada com melhores taxas de sobrevida. Por outro lado, a presença de invasão vascular, apesar do tamanho pequeno das lesões, é um fator associado a péssimo prognóstico. / Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Liver cirrhosis is the major risk factor for this tumor. Screening for HCC in patients with cirrhosis has been recommended, in the belief that detection and treatment of early HCC improves patient survival. The aims of this study were to analyze the overall survival of small HCC in cirrhotic patients and identify independent predictors of survival, in Brazil. Methods: Between January 1998 and December 2003, seventy-four cirrhotic patients with hepatocellular carcinoma were evaluated satisfying the following criteria: HCC of 30 mm or smaller and a maximum of three lesions. Predictors of survival were identified using the Kaplan-Meier and the Cox model. Results: Mean age was 58 years-old (32-77), 71% of patients was male, 64% had hepatitis C, 60% were Child-Pugh A, mean MELD score was 11 and 79% had portal hypertension. At the time of diagnosis, 71% had one tumor, the size of the main tumor was smaller than 20 mm in 47%, mean AFP level was 131 ng/ml. Three patients had portal vein thrombosis, suggesting vascular invasion. Fifty patients (67.5%) were included in the liver transplant list, but it was performed in only four patients. Tumor resection was possible in four patients. Forty-eight (64.8%) patients received percutaneous treatment (radiofrequency ablation or percutaneous ethanol injection). Nine patients did not receive any cancer treatment. The overall survival rates were 80%, 62%, 41% and 17% at 12, 24, 36 and 60 months, respectively. The mean length of follow-up after HCC diagnosis was 23 months (median 22 months, range 1-86 months) for the entire group. During follow-up a total of 39 deaths related to liver failure or HCC progression occurred. Univariate analysis of the 74 patients showed that MELD score greater than 11 (p = 0.016), Child-Pugh classification (p = 0.007), AFP > 100 ng/ml (p = 0.006), more than one lesion (p = 0.041), tumor diameter > 20 mm (p = 0.009) and presence of vascular invasion (p < 0.0001) were significant predictors of survival. Cox regression analysis identified vascular invasion (RR = 14.60 - IC 95% = 3.3 - 64.56 - p < 0.001) and tumor size > 20mm (RR = 2.14 - IC 95% = 1.07 - 4.2 - p = 0.030) as independent predictors of decreased survival. Treatment of HCC was related to increased overall survival. Conclusion: Identification of small tumors of up to 20 mm diameter is related to increase survival. Nevertheless, vascular invasion, in spite of the small diameter of the lesions, is a factor associated with dismal prognosis.
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Variations of the coeliac artery and hepatic artery origins and their importance in selective internal radiation therapy.January 1998 (has links)
by Ho Wai-chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references. / Abstract also in Chinese. / Title / Dedication / Abstract --- p.i / Table of Contents --- p.iv / Glossary of abbreviation used in the thesis --- p.vi / List of figures --- p.viii / List of tables --- p.xvii / Acknowledgement --- p.xix / Statement of Originality --- p.xx / Chapter Chapter 1 ...... --- Introduction --- p.14 / Chapter Chapter 2...... --- Basic Principle / Chapter 2.1 --- The liver - a vital organ --- p.2-1 / Chapter 2.2 --- Blood supply to the liver --- p.2-7 / Chapter 2.3 --- Normal arterial anatomy of the coeliac axis --- p.2-11 / Chapter 2.4 --- Common anomalies of the coeliac axis --- p.2-17 / Chapter 2.5 --- Previous classification of coeliac anomaies --- p.2-24 / Chapter 2.6 --- Knowledge of arterial anomaly in relation to surgery --- p.2-31 / Chapter 2.7 --- Trans-catheter treatment of hepatocellular carcinoma --- p.2-33 / Chapter 2.8 --- Prevalence of hepatocellular carcinoma in H.K Chinese --- p.2-42 / Chapter 2.9 --- Management of hepatocellular carcinoma in Hong Kong --- p.2-43 / Chapter Chapter 3...... --- Definitions --- p.3-1 / Chapter Chapter 4...... --- Objectives of the study --- p.4-1 / Chapter Chapter 5...... --- "Materials, methods and subjects" / Chapter 5.1 --- Materials --- p.5-1 / Chapter 5.2 --- Methods --- p.5-3 / Chapter 5.3 --- Subjects --- p.5-10 / Chapter Chapter 6...... --- Results / Chapter 6.1 --- Coeliac axis --- p.6-5 / Chapter 6.2 --- Common hepatic artery --- p.6-9 / Chapter 6.3 --- Proper hepatic artery --- p.6-11 / Chapter 6.4 --- Right hepatic artery --- p.6-12 / Chapter 6.5 --- Middle hepatic artery --- p.6-20 / Chapter 6.6 --- Left hepatic artery --- p.6-28 / Chapter 6.7 --- Gastroduodenal artery --- p.6-33 / Chapter 6.8 --- Right gastric artery --- p.6-37 / Chapter 6.9 --- Left gastric artery --- p.5-45 / Chapter 6.10 --- Splenic artery --- p.6-49 / Chapter 6.11 --- Summary of results --- p.6-51 / Chapter Chapter 7...... --- Discussion / Chapter 7.1 --- Introduction --- p.74 / Chapter 7.2 --- Selective Internal Radiation --- p.7-3 / Chapter 7.3 --- Coeliac axis --- p.7-10 / Chapter 7.4 --- Common hepatic & proper hepatic artery --- p.7-7 / Chapter 7.5 --- Right hepatic artery --- p.7-14 / Chapter 7.6 --- Middle hepatic artery --- p.7-18 / Chapter 7.7 --- Left hepatic artery --- p.7-25 / Chapter 7.8 --- Gastroduodenal artery --- p.7-30 / Chapter 7.9 --- Right gastric artery --- p.7-35 / Chapter 7.10 --- Left gastric artery --- p.7-43 / Chapter 7.11 --- Splenic artery --- p.7-45 / Chapter 7.12 --- Comparison with the golden classics --- p.7-47 / Chapter 7.13 --- Comparison of subjects with HCC & without HCC --- p.7-50 / Chapter 7.14 --- Comparison of the male group and the female group --- p.7-51 / Chapter Chapter 8...... --- Conclusions / References --- p.B-1 / Bibliography --- p.B-1 / Appendix I Schematic diagram of histological anatomy of the liver --- p.A-l / Appendix II Embryology --- p.A-2 / "Appendix III Percentages of occurrence of the different types of coeliac axis, by Michels' study" --- p.A-3 / "Appendix IV Percentages of occurrence of the different types of the hepatic arterial blood supply, by Michels' study" --- p.A-4 / Appendix V No. of deaths from malignant liver cancer in Hong Kong froml984 to1993 --- p.A-5 / Appendix VI Flow chart for HCC management in PWH of Hong Kong --- p.A-6 / Appendix VII Comparison with Michels' study --- p.A-7 / Appendix VIII Comparison of the group with HCC and the group without HCC --- p.A-8 / Appendix IX Comparison of the male and female group --- p.A-9
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Expression, sequencing and transfection studies of the hepatitis B virus x gene from human hepatocellular carcinoma tissues.January 2000 (has links)
Chan Ming Lok. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 93-108). / Abstracts in English and Chinese. / Ackowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Chapter Chapter 1 --- Introduction and Objectives / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Geographical Distribution --- p.1 / Chapter 1.1.3 --- Sex and Age --- p.1 / Chapter 1.1.4 --- Etiology --- p.2 / Chapter 1.1.5 --- Molecular Basis of HCC --- p.3 / Chapter 1.1.6 --- Situation in China and Hong Kong --- p.4 / Chapter 1.2 --- The Hepatitis B Virus --- p.5 / Chapter 1.2.1 --- Morphology --- p.5 / Chapter 1.2.2 --- Structure of the HBV Genome --- p.6 / Chapter 1.2.3 --- Functional Domains of the HBV Genome --- p.9 / Chapter 1.2.4 --- Pathogenesis of HBV Infection --- p.11 / Chapter 1.3 --- HBx --- p.12 / Chapter 1.3.1 --- The HBV x Gene --- p.12 / Chapter 1.3.2 --- The HBX Protein --- p.13 / Chapter 1.3.3 --- "Preferential HBX Expression in Sera, Hepatitis, Cirrhosis and HCC" --- p.13 / Chapter 1.3.4 --- Cellular Localization of HBX --- p.14 / Chapter 1.3.5 --- Animal Studies --- p.15 / Chapter 1.3.6 --- Functional Studies on HBX --- p.15 / Chapter 1.3.7 --- Variations in the HBx Gene --- p.21 / Chapter 1.4 --- Objectives of this Study --- p.24 / Chapter Chapter 2 --- Methods and Materials Methods / Chapter 2.1 --- Paraffin Embedding of Patient Tissue Samples --- p.26 / Chapter 2.1.1 --- Tissue Processing --- p.26 / Chapter 2.1.2 --- Paraffin Embedding of Tissue Samples --- p.26 / Chapter 2.2 --- Sectioning of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3 --- Immunohistochemical Staining of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3.1 --- Dewaxing of Paraffin-Embedded Tissue Sections --- p.26 / Chapter 2.3.2 --- Rehydration of Tissue Sections --- p.27 / Chapter 2.3.3 --- Antigen Retrieval --- p.27 / Chapter 2.3.4 --- Quenching of Endogenous Hydrogen Peroxidase --- p.27 / Chapter 2.3.5 --- Blocking of Endogenous Biotin and Non-Specific Protein Binding --- p.27 / Chapter 2.3.6 --- Antibody Incubation and Color Development --- p.27 / Chapter 2.3.7 --- Counterstaining and Coverslip Mounting --- p.28 / Chapter 2.3.8 --- Interpretation of Immunostaining Results --- p.28 / Chapter 2.4 --- DNA Extraction from HCC Tissues --- p.28 / Chapter 2.4.1 --- Sectioning of Frozen HCC Specimens --- p.28 / Chapter 2.4.2 --- Proteinase K Digestion and Phenol Chloroform Extraction --- p.29 / Chapter 2.4.3 --- Ethanol Precipitation and Re-suspension in Tris-EDTA (TE) Buffer --- p.29 / Chapter 2.5 --- Quantitation and Purity Check of Extracted DNA --- p.29 / Chapter 2.6 --- Quality Check for Extracted Genomic DNA --- p.30 / Chapter 2.6.1 --- Agarose Gel Electrophoresis --- p.30 / Chapter 2.6.2 --- Polymerase Chain Reaction (PCR) of the β-globin Gene --- p.30 / Chapter 2.6.3 --- Analysis of PCR Fragments by Agarose Gel Electrophoresis --- p.30 / Chapter 2.7 --- Polymerase Chain Reaction Amplification of HBs and HBx Genes of the Hepatitis B Virus --- p.31 / Chapter 2.8 --- Southern Blot of HBx PCR Fragments --- p.31 / Chapter 2.8.1 --- Immobilization of DNA onto a Positively Charged Nylon Membrane and Pre-hybridization --- p.31 / Chapter 2.8.2 --- Radio-labeling of an HBV Probe --- p.32 / Chapter 2.8.3 --- Hybridization of a 32P-labeled HBV Probe and Film Exposure --- p.32 / Chapter 2.9 --- Cloning of PCR Fragments into pGEM®-T Vector for Sequencing --- p.33 / Chapter 2.9.1 --- Gel Extraction and Purification --- p.33 / Chapter 2.9.2 --- Ligation --- p.33 / Chapter 2.10 --- Transformation of Competent DH5a cells --- p.34 / Chapter 2.10.1 --- Preparation of Competent DH5α Using Calcium Chloride --- p.34 / Chapter 2.10.2 --- Heat Shock of Competent DH5α Cells --- p.34 / Chapter 2.10.3 --- Plating of Transformed Cells onto LB Agar Plates --- p.34 / Chapter 2.10.4 --- Screening of Transformants for Inserts --- p.35 / Chapter 2.11 --- Miniprep of Plasmid DNA --- p.35 / Chapter 2.11.1 --- Inoculation of Bacterial Clones --- p.35 / Chapter 2.11.2 --- DNA Extraction by Alkaline Lysis and Phenol/Chloroform --- p.35 / Chapter 2.11.3 --- Ethanol Precipitation and Re-suspension in TE Buffer --- p.35 / Chapter 2.11.4 --- Confirmation of Positive Clones --- p.36 / Chapter 2.12 --- Sequencing of pGEM®-T Cloned HBx PCR Fragments --- p.36 / Chapter 2.13 --- Construction of the HBx-GFP Plasmid --- p.36 / Chapter 2.13.1 --- PCR Amplification of HBx Gene Inserts --- p.36 / Chapter 2.13.2 --- Confirmation of HBx Insert Sequence by DNA Sequencing --- p.37 / Chapter 2.13.3 --- Restriction Digest of HBx-pGEM®-T Plasmids to Obtain HBx Inserts --- p.37 / Chapter 2.13.4 --- Restriction Digest of pEGFP-Nl Cloning Vector for Cloning --- p.37 / Chapter 2.13.5 --- Ligation of HBx Inserts into the pEGFP Cloning Vector --- p.37 / Chapter 2.14 --- Large Scale Plasmid DNA Preparation --- p.38 / Chapter 2.15 --- Cell Culture --- p.39 / Chapter 2.16 --- Transfection using LipofectAminéёØ --- p.39 / Chapter 2.16.1 --- Seeding of Cells for Coverslip Growth --- p.39 / Chapter 2.16.2 --- Transfection using LipofecAminéёØ --- p.39 / Chapter 2.17 --- Cell Fixation and DAPI Staining Materials --- p.40 / Chapter 2.18 --- Chemicals --- p.41 / Chapter 2.19 --- Antibodies --- p.41 / Chapter 2.20 --- "Formalin-fixed, Paraffin Embedded Tissues of HCC Tissues from Xiamen" --- p.41 / Chapter 2.21 --- Frozen Liver Tissues --- p.41 / Chapter 2.22 --- PCR Reagents --- p.43 / Chapter 2.23 --- Primers --- p.43 / Chapter 2.24 --- Plasmid --- p.43 / Chapter 2.25 --- Enzymes --- p.43 / Chapter 2.26 --- Ligation Reagents --- p.43 / Chapter 2.27 --- Cloning Vectors --- p.45 / Chapter 2.28 --- Competent Cell --- p.45 / Chapter 2.29 --- Hela and HepG2 Cell Line --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Hepatitis B Virus Status of HCC Patients from Hong Kong and Xiamen --- p.46 / Chapter 3.2 --- Immunohistochemical Studies of the HBx Protein in Hong Kong and Xiamen HCC --- p.46 / Chapter 3.2.1 --- Cross Reaction of Anti-99 with Cytokeratin 18 (CK18) --- p.46 / Chapter 3.2.2 --- HBx Expression in HCC Patient Tissue Samples from Hong Kong --- p.50 / Chapter 3.2.3 --- HBxAg Staining in HCC Tissue Samples from Xiamen --- p.50 / Chapter 3.3 --- Agarose Gel Electrophoresis of DNA Extracted from Frozen Liver Tissues --- p.50 / Chapter 3.4 --- PCR Amplification of the β-globin Gene --- p.55 / Chapter 3.5 --- PCR Amplification of the HBs Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.6 --- PCR Amplification of the HBx Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.7 --- Amplification of the HBx Gene from Serum Samples of Chronic Hepatitis B Virus from Hong Kong Using Nested PCR --- p.61 / Chapter 3.8 --- Southern Blot of HBx PCR Fragments --- p.61 / Chapter 3.9 --- Cloning and Sequencing of the HBx Gene in HCC and Chronic Hepatitis B Patient Samples from Hong Kong --- p.61 / Chapter 3.10 --- Expression Pattern of Wild-type HBx-GFP Fusion Protein in Transiently Transfected HeLa and HepG2 Cells --- p.73 / Chapter 3.11 --- Expression Patterns of HBx-GFP with and without Mutations at Codons 130 and 131 in HeLa and HepG2 Cell Line --- p.78 / Chapter 3.12 --- Growth Kinetics of HeLa Cells Transfected with GFP and Wild-type HBx-GFP with and without Mutations in Codons 130 and131 --- p.81 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- HBxAg Expression in Tumorous and Surrounding Non-tumorous Tissues --- p.83 / Chapter 4.2 --- "Detection of the HBx Gene in Sera, Non-tumorous and Tumorous Tissues" --- p.84 / Chapter 4.3 --- HBx Gene Mutations in Chronic Hepatitis and HCC --- p.85 / Chapter 4.3.1 --- Codon 127 (HBV nt 1752-1754) --- p.85 / Chapter 4.3.2 --- Codons 130 and 131 (HBV nt 1761-1766) --- p.86 / Chapter 4.3.3 --- Lack of Correlation between HBx Gene Mutations and Lack of HBxAg Expression --- p.87 / Chapter 4.4 --- Cellular Localization of HBxAg in Transiently Transfected Cells Lines --- p.88 / Chapter 4.5 --- Functional Difference Between Wild-type and Mutant HBX Protein --- p.89 / Chapter Chapter 5 --- Conclusions and Directions for Further Studies / Chapter 5.1 --- Conclusions --- p.91 / Chapter 5.2 --- Directions for Further Studies --- p.92 / References --- p.93 / Appendix / Chapter A1 --- Recipes of Reagents Used in this Study --- p.109 / Chapter A2 --- Schematic Setup of Downward Capillary Transfer of DNA --- p.112 / Chapter A3 --- Circle Map of the pGEM®-T Cloning Vector and Construct of the HBx-pGEM®-T Plasmid --- p.113 / Chapter A4 --- Circle Map of the pEGFP-Nl Cloning Vector and Construct of the HBx-GFP Plasmid --- p.114
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Effects of arsenic trioxide on human hepatoma cells.January 2001 (has links)
Siu Pak-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-174). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Contents --- p.vi / List of Figures and Tables --- p.xiii / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Characteristics of Arsenic Compound --- p.1 / Chapter 1.1.1 --- Arsenic Compounds are Used as Poison --- p.1 / Chapter 1.1.2 --- Arsenic Compounds are Used as Medicine --- p.2 / Chapter 1.2 --- Arsenic Trioxide is a Traditional Chinese Medicine --- p.3 / Chapter 1.3 --- Properties of Arsenic Trioxide --- p.5 / Chapter 1.4 --- Use of Arsenic Trioxide in Cancer Treatment --- p.7 / Chapter 1.4.1 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Acute Promyelocytic Leukemia --- p.7 / Chapter 1.4.1.1 --- Characteristics of Acute Promyelocytic Leukemia --- p.7 / Chapter 1.4.1.2 --- Treatment of Acute Promyelocytic Leukemia with All-Trans Retinoic Acid --- p.10 / Chapter 1.4.1.3 --- Treatment of Acute Promyelocytic Leukemia with Arsenic Trioxide --- p.11 / Chapter 1.4.1.4 --- Action Mechanism of Arsenic Trioxide --- p.13 / Chapter 1.4.2 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Non-APL Leukemia --- p.15 / Chapter 1.4.3 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Solid Tumors --- p.16 / Chapter 1.5 --- Human Hepatocellular Carcinoma --- p.16 / Chapter 1.5.1 --- The Incidence of Liver Cancer --- p.16 / Chapter 1.5.2 --- Classification of Liver Cancer --- p.17 / Chapter 1.6 --- Aim of the Project --- p.17 / Chapter 1.6.1 --- In Vitro Study of the Effect of Arsenic Trioxide on HepG2 Cells --- p.19 / Chapter 1.6.2 --- In Vivo Study of the Effect of Arsenic Trioxide by Tumor-Bearing Nude Mice Model --- p.20 / Chapter 1.6.3 --- "In Vitro Study of the Effect of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" --- p.22 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Cell Lines and Culture Medium --- p.24 / Chapter 2.1.1.1 --- Cell Lines --- p.24 / Chapter 2.1.1.2 --- Culture Medium --- p.25 / Chapter 2.1.2 --- Chemicals --- p.26 / Chapter 2.1.3 --- Reagents and Buffers --- p.27 / Chapter 2.1.3.1 --- Phosphate Buffered Saline (PBS) --- p.27 / Chapter 2.1.3.2 --- "3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) Solution" --- p.27 / Chapter 2.1.3.3 --- Reagents for DNA Fragmentation Assay --- p.21 / Chapter 2.1.3.3.1 --- DNA Lysis Buffer --- p.27 / Chapter 2.1.3.3.2 --- Tris-EDTA (TE) Buffer --- p.27 / Chapter 2.1.3.3.3 --- Tris-Acetate (TAE) Buffer --- p.28 / Chapter 2.1.3.3.4 --- Proteinase K and Ribonuclease A (RNase A) --- p.28 / Chapter 2.1.3.3.5 --- 6X DNA Loading Dye --- p.28 / Chapter 2.1.3.3.6 --- One Hundred Base-Pair DNA Ladder --- p.28 / Chapter 2.1.3.4 --- Reagents for Western Blot Analysis --- p.29 / Chapter 2.1.3.4.1 --- SDS Lysis Buffer --- p.29 / Chapter 2.1.3.4.2 --- 4X Lower Gel Buffer --- p.29 / Chapter 2.1.3.4.3 --- 4X Upper Gel Buffer --- p.29 / Chapter 2.1.3.4.4 --- 10X SDS Running Buffer --- p.29 / Chapter 2.1.3.4.5 --- 2X SDS Sample Loading Dye --- p.30 / Chapter 2.1.3.4.6 --- Electroblotting Buffer --- p.30 / Chapter 2.1.3.4.7 --- Tris-Buffered Saline with 01% Tween-20 (TBS-T) --- p.30 / Chapter 2.1.3.4.8 --- Lysis Buffer for Detection of the Release of Cytochrome C --- p.31 / Chapter 2.1.3.5 --- Propidium Iodide (PI) --- p.31 / Chapter 2.1.3.6 --- "5,5 ´ة,6,6´ة-tetrachloro-1,1',3,3 '-tetraethylbenzimidazolyl carbocyanine Iodide (JC-1)" --- p.31 / Chapter 2.1.3.7 --- Reagents for In Vivo Study --- p.32 / Chapter 2.1.3.7.1 --- Saline --- p.32 / Chapter 2.1.3.7.2 --- Homogenizing Buffer --- p.32 / Chapter 2.1.3.7.3 --- 10% Buffered Formalin --- p.32 / Chapter 2.1.3.7.4 --- Acid Alcohol --- p.32 / Chapter 2.1.3.7.5 --- Scott's Tap Water --- p.32 / Chapter 2.1.3.7.6 --- 0.5% Aqueous Eosin --- p.33 / Chapter 2.2 --- Methods --- p.33 / Chapter 2.2.1 --- MTT Assay --- p.33 / Chapter 2.2.2 --- Trypan Blue Exclusion Assay --- p.34 / Chapter 2.2.3 --- Analysis of Cell-Cycle Phase Distribution by Flow Cytometry with PI Staining --- p.34 / Chapter 2.2.4 --- DNA Fragmentation Assay --- p.35 / Chapter 2.2.5 --- Quantification of Apoptosis by Flow Cytometry with Annexin V-PI Staining --- p.36 / Chapter 2.2.6 --- Assessment of the Change in Mitochondrial Membrane Potential (ΔΦm) --- p.37 / Chapter 2.2.7 --- Western Analysis --- p.38 / Chapter 2.2.8 --- Glucose Uptake Assay --- p.40 / Chapter 2.2.9 --- ATP Production Assay --- p.41 / Chapter 2.2.10 --- In Vivo Study --- p.44 / Chapter 2.2.10.1 --- Animal Model --- p.44 / Chapter 2.2.10.2 --- Cell Line --- p.44 / Chapter 2.2.10.3 --- Treatment with Arsenic Trioxide --- p.44 / Chapter 2.2.10.4 --- Assessment of the Anti-Cancer Activity of Arsenic Trioxide --- p.45 / Chapter 2.2.10.5 --- Tissue Sample Preparation --- p.45 / Chapter 2.2.10.5.1 --- Preparation of Plasma --- p.45 / Chapter 2.2.10.5.2 --- Preparation of Liver Tissue Homogenate --- p.46 / Chapter 2.2.10.5.3 --- Preparation of Cytosolic Fraction --- p.46 / Chapter 2.2.10.6 --- Measurement of the Plasma Enzyme Activity --- p.46 / Chapter 2.2.10.6.1 --- "Plasma Creatine Kinase (CK) Activity, Plasma Lactate Dehydrogenase (LDH) Activity, Plasma Alanine Transaminase (ALT) Activity and Plasma Asparate Transaminase (AST) Activity" --- p.46 / Chapter 2.2.10.7 --- Preparation of Tissue for Light Microscopic Study --- p.48 / Chapter 2.2.10.8 --- Measurement of the Basal Reduced Glutathione (GSH) Level of Liver Tissue --- p.51 / Chapter 2.2.10.9 --- "Measurement of the Activity of Antioxidant Enzyme, Glutathione S-Transferase (GST) of Liver Tissue" --- p.53 / Chapter 2.3 --- Statistical Analysis --- p.54 / Chapter Chapter 3 --- "In Vitro Study of Arsenic Trioxide on Acute Promyelocytic Leukemia Cell Line, NB-4" / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Principle of Flow Cytometry with Annexin V-PI Staining --- p.56 / Chapter 3.3 --- The Effect of Arsenic Trioxide on Cell Proliferation of NB-4 Cells --- p.59 / Chapter 3.4 --- Study of the Action Mechanism of Arsenic Trioxide upon Treatment of NB-4 Cells --- p.61 / Chapter 3.5 --- Summary --- p.63 / Chapter Chapter 4 --- "In Vitro Study of Arsenic Trioxide on Human Hepatocellular Carcinoma Cell Line, HepG2" / Chapter 4.1 --- Introduction --- p.64 / Chapter 4.2 --- The Effect of Arsenic Trioxide on Cell Proliferation of HepG2 Cells by MTT Assay --- p.66 / Chapter 4.3 --- The Effect of Arsenic Trioxide on HepG2 Cells at Clinically Achievable Concentration --- p.68 / Chapter 4.3.1 --- The Cytotoxicity of Arsenic Trioxide on HepG2 Cells by Trypan Blue Exclusion Assay --- p.68 / Chapter 4.3.2 --- The Effect of Arsenic Trioxide on Cell-Cycle Phase Distribution --- p.71 / Chapter 4.3.3 --- The Underlying Mechanism of the Cytotoxic Effect of Arsenic Trioxide 一 Necrosis or Apoptosis? --- p.74 / Chapter 4.3.3.1 --- DNA Fragmentation Assay --- p.74 / Chapter 4.3.3.2 --- Flow Cytometry with Annexin V-PI Staining --- p.76 / Chapter 4.3.3.3 --- Brief Conclusion --- p.78 / Chapter 4.3.4 --- The Study of the Mechanism of Apoptotic Pathway --- p.78 / Chapter 4.3.4.1 --- Activation of Caspase-3 upon Arsenic Trioxide Treatment --- p.79 / Chapter 4.3.4.2 --- The Participation of Mitochondria in Arsenic Trioxide-Induced Apoptosis --- p.81 / Chapter 4.3.4.2.1 --- The Change in Mitochondrial Membrane Potential upon Arsenic Trioxide Treatment --- p.81 / Chapter 4.3.4.2.2 --- The Study of the Release of Cytochrome C from the Mitochondria to Cytosol upon Treatment with Arsenic Trioxide --- p.85 / Chapter 4.3.4.2.3 --- Brief Conclusion --- p.87 / Chapter 4.4 --- Arsenic Trioxide Mediated Its Effect via Other Action Mechanisms --- p.87 / Chapter 4.4.1 --- The Effect of Arsenic Trioxide on the Expression of Glucose Transporters 1 and2 --- p.88 / Chapter 4.4.2 --- The Effect of Arsenic Trioxide on Glucose Uptake --- p.91 / Chapter 4.4.3 --- The Effect of Arsenic Trioxide on ATP Production --- p.93 / Chapter 4.4.4 --- Brief Conclusion --- p.93 / Chapter 4.5 --- Summary --- p.95 / Chapter Chapter 5 --- In Vivo Study of Arsenic Trioxide on HepG2-Bearing Nude Mice / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Treatment with Arsenic Trioxide --- p.97 / Chapter 5.3 --- Assessment of the Anti-Tumor Effect of Arsenic Trioxide --- p.99 / Chapter 5.4 --- The Effect of Arsenic Trioxide toward Normal Tissues --- p.103 / Chapter 5.4.1 --- The Effect of Arsenic Trioxide on Liver --- p.104 / Chapter 5.4.1.1 --- Morphological Study --- p.104 / Chapter 5.4.1.2 --- Enzymatic Study --- p.107 / Chapter 5.4.1.3 --- Brief Conclusion --- p.107 / Chapter 5.4.2 --- The Effect of Arsenic Trioxide on Heart --- p.110 / Chapter 5.4.2.1 --- Morphological Study --- p.110 / Chapter 5.4.2.2 --- Enzymatic Study --- p.112 / Chapter 5.4.2.3 --- Brief Conclusion --- p.112 / Chapter 5.5 --- Involvement of the Glutathione Redox System --- p.115 / Chapter 5.5.1 --- Basal GSH Level --- p.115 / Chapter 5.5.2 --- The Activity of Glutathion S-Transferase --- p.117 / Chapter 5.5.3 --- Brief Conclusion --- p.117 / Chapter 5.6 --- Summary --- p.120 / Chapter Chapter 6 --- "In Vitro Study of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" / Chapter 6.1 --- Introduction --- p.121 / Chapter 6.2 --- The Effect of Doxorubicin on the Parental HepG2 Cells and R-HepG2 Cells by MTT Assay --- p.123 / Chapter 6.3 --- The Effect of Arsenic Trioxide on Cell Proliferation of R-HepG2 Cells by MTT Assay --- p.126 / Chapter 6.4 --- The Effect of Arsenic Trioxide on Cell-Cycle Phase Distribution of R-HepG2 Cells --- p.129 / Chapter 6.5 --- Trioxide on R-HepG2 Cells ´ؤ Necrosis or Apoptosis? --- p.131 / Chapter 6.5.1 --- DNA Fragmentation Assay --- p.131 / Chapter 6.5.2 --- Flow Cytometry with Annexin V-PI Staining --- p.133 / Chapter 6.5.3 --- Brief Conclusion --- p.133 / Chapter 6.6 --- Examination of the Probable Involvement of Arsenic Trioxide as a Substrate of P-Glycoprotein --- p.135 / Chapter 6.7 --- Summary --- p.137 / Chapter Chapter 7 --- Discussion / Chapter 7.1 --- The Significance of the Study of Arsenic Trioxide in the Treatment of Arsenic Trioxide --- p.138 / Chapter 7.2 --- Comparison of Preparation of Drug in Present Study with Others --- p.140 / Chapter 7.3 --- Effect of Arsenic Trioxide on Human Hepatocellular Carcinoma --- p.142 / Chapter 7.4 --- Mechanism Study of Arsenic Trioxide --- p.142 / Chapter 7.5 --- Dosage of Arsenic Trioxide Used in In Vivo Study --- p.152 / Chapter 7.6 --- Cytotoxicity of Arsenic Trioxide toward Normal Tissues --- p.153 / Chapter 7.7 --- "Effect of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" --- p.154 / Chapter 7.8 --- Conclusions and Future Prospect --- p.156 / Chapter Chapter 8 --- References / Chapter 8.1 --- English References --- p.158 / Chapter 8.2 --- Chinese References --- p.174 / Chapter 8.3 --- Online References --- p.174
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Role of polymeric immunoglobulin receptor in pancreatic ductal adenocarcinomaArumugam, Prabhu January 2017 (has links)
Introduction: Polymeric immunoglobulin receptor (pIgR) traffics Immunoglobulins (IgA and IgM) through epithelial cells in normal mucosae but neither are expressed in the normal pancreas. Recent work has demonstrated pIgR to be upregulated in hepatocellular carcinoma, even though it is not expressed in normal liver cells. High pIgR levels are associated with poor survival and distant metastases for a number of cancers such as nasopharyngeal cancers, lung and oesophageal cancers. Recent work from our laboratory suggested pIgR may be upregulated in pancreatic ductal adenocarcinoma (PDAC). My aim was to assess pIgR's role in PDAC by interrogating human PDAC tissue samples as well using cell biology experimental tools. Methods: pIgR expression was manipulated (siRNA and shRNA) in cell lines to evaluate its subsequent effect on cell behaviour in 2D assays as well as 3D organotypics models. Tissue Microarrays of patients with PDAC were analysed after pIgR, αSMA, E-Cadherin and Picrosirius Red staining to assess their role as a combined bio-marker panel. Results: Cytokines such as interleukin 4 (IL4) and Tumour Necrosis Factor (TNFα) could not modulate pIgR expression in PDAC cell lines despite this effect being seen in other studies using colorectal and nasopharyngeal cancer cell lines. Downregulation in pIgR expression in Capan1 cell line resulted in reduction of cellular proliferation (n= 3, P < 0.05, Friedman test), adhesion (n= 3, P < 0.05, Kruskal-Wallis) and migration (n= 3, P < 0.05, Kruskal-Wallis). In 3D organotypic models, pIgR downregulation resulted in reduced cancer cell invasion (n= 9, P < 0.05, Kruskal- Wallis) and diminished contraction of gels (n= 9, P < 0.05, Kruskal-Wallis). In human PDAC, decreased E-cadherin expression correlates with increased pIgR expression through pancreatic intra-epithelial neoplasia (PanIN) progression. There was no IgA expression in PDAC. pIgR expression had no clinical correlation with routine prognostic measures such as differentiation, lymph node metastasis (n= 88, P=0.5012, Kruskal-Wallis). Even in combination with stromal indices (α-smooth muscle action (SMA) and Picrosirius red), low pIgR scores had no statistically significant impact on prognosis but had a trend towards better survival (n= 88, P=0.2791, Mann-Whitney U test). Conclusion: pIgR may be involved in progression from pre-neoplastic lesions such as PanIN to PDAC. pIgR may have a biological impact on cellular motility and invasion due to yet to be deciphered signalling cascades with marked effect on cellular phenotype. Careful analysis is required to study the impact of pIgR on prognostic impact bearing in mind the histological sub-types of pancreatic cancer.
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Harnessing the immune response to optimise treatment strategies in chronic hepatitis BGill, Upkar S. January 2018 (has links)
Chronic Hepatitis B (CHB) related cirrhosis and hepatocellular carcinoma (HCC) account for more than 750,000 deaths per year. Current therapies for CHB are limited in achieving HBsAg decline/loss and thus there remains a pressing need for curative treatment strategies. Although, Pegylated Interferon-α (Peg-IFNα) may be used, the majority of patients progress to nucleos(t)ide analogue (NUC) therapy due to treatment failure. Peg-IFNα and NUCs used in isolation act differentially on the immune response; Peg-IFNα induces NK cell activation and NUC therapy may partially restore T cell function. NK cells are important antiviral effectors, highly enriched in the liver, with the potential to regulate immunopathogenesis in persistent viral infections. Here we examined the NK cell pool in HBeAg-positive CHB patients treated with Peg-IFNα and whether changes in the NK cell repertoire are induced when patients are 'primed' with Peg-IFNα and importantly, whether these changes are sustained or further modulated long-term after switching to sequential NUC therapy. The cumulative expansion of CD56bright NK cells driven by 48-weeks of Peg-IFNα was maintained at higher than baseline levels throughout the subsequent 9 months of sequential NUCs. Peg- IFNα-expanded NK cells showed further augmentation in their expression of the activating NK cell receptors during sequential NUCs. The expansion in proliferating, functional NK cells and HBsAg reduction was greater and more pronounced following sequential NUCs than in patients treated with de novo NUCs. This highlights the potential benefit of Peg-IFNα- priming, providing mechanistic insights for the further optimisation of treatment strategies to achieve sustained responses. Sustained boosting of NK cells on sequential NUCs following Peg-IFNα-priming has not previously been described raising the potential of 'long-lived' NK cell populations in keeping with their emerging adaptive features. These findings provide a mechanistic and immunological rationale to explore combination/sequential treatment strategies for CHB, including on-treatment immune responses in the liver, whilst awaiting the emergence of new therapies in the field.
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Effects of polyphyllin D on the induction of apoptosis in human hepatocellular carcinoma HepG2 cells and its multidrug resistant derivative RHepG2 cells.January 2004 (has links)
Ong Chik Ying Rose. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 181-195). / Abstracts in English and Chinese. / Acknoledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.iii / Abstract in Chinese --- p.v / List of Publications and Abstracts --- p.vii / List of Figures --- p.viii / List of Tables --- p.xi / Contents --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Saponins --- p.3 / Chapter 1.1.1 --- Structure of saponins --- p.3 / Chapter 1.1.2 --- Occurrence of saponins --- p.7 / Chapter 1.1.3 --- Bioactivities of saponins --- p.8 / Chapter 1.2 --- Paris Polyphylla --- p.9 / Chapter 1.2.1 --- Chonglou --- p.11 / Chapter 1.3 --- Polyphyllin D --- p.12 / Chapter 1.4 --- Apoptosis --- p.15 / Chapter 1.4.1 --- Apoptosis and necrosis --- p.16 / Chapter 1.4.2 --- Initiation phase of apoptosis --- p.16 / Chapter 1.4.2.1 --- Extrinsic apoptotic pathway --- p.18 / Chapter 1.4.2.2 --- Intrinsic apoptotic pathway --- p.19 / Chapter 1.4.3 --- Execution phase of apoptosis --- p.19 / Chapter 1.4.4 --- Termination phase of apoptosis --- p.20 / Chapter 1.5 --- Multi-drug resistance (MDR) --- p.21 / Chapter 1.5.1 --- MDR mediated by decreased drug accumulation --- p.21 / Chapter 1.5.2 --- MDR mediated by enhanced anti-oxidant enzyme activities --- p.25 / Chapter 1.5.3 --- MDR mediated by enhanced detoxification of drugs --- p.25 / Chapter 1.5.4 --- MDR mediated by enhanced DNA repair system --- p.26 / Chapter 1.5.5 --- MDR mediated by altered apoptotic pathway --- p.26 / Chapter 1.5.6 --- Current strategies for overcoming multidrug resistance in cancer --- p.27 / Chapter 1.6 --- Hepatocellular carcinoma (HCC) --- p.30 / Chapter 1.7 --- Objectives of project --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Culture of Cells --- p.34 / Chapter 2.1.1.1 --- Cell lines --- p.34 / Chapter 2.1.1.2 --- Preservation of Cells --- p.35 / Chapter 2.1.2 --- Culture Media --- p.36 / Chapter 2.1.2.1 --- RPMI 1640 (Phenol Red Medium) --- p.36 / Chapter 2.1.2.2 --- RPMI 1640 (Phenol Red-free Medium) --- p.36 / Chapter 2.1.3 --- Buffers and Reagents --- p.37 / Chapter 2.1.3.1 --- Buffer for Common Use --- p.37 / Chapter 2.1.3.2 --- Reagents for Annexin-V-FITC/PI Assay --- p.37 / Chapter 2.1.3.3 --- Reagents for Western Blotting Analysis --- p.37 / Chapter 2.1.4 --- Chemicals --- p.40 / Chapter 2.1.4.1 --- Fluorescence Dyes --- p.40 / Chapter 2.1.4.2 --- Antibodies --- p.41 / Chapter 2.1.4.3 --- Other Chemicals --- p.42 / Chapter 2.1.5 --- Summary of chemicals used in this study --- p.43 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- In vitro Cell Cytotoxicity Assay --- p.48 / Chapter 2.2.1.1 --- AlamarBlue Assay --- p.48 / Chapter 2.2.2 --- Flow Cytometry --- p.50 / Chapter 2.2.2.1 --- Analysis by Flow Cytometry --- p.50 / Chapter 2.2.2.2 --- Determination of Apoptotic and Late Apoptotic/Necrotic Cells with Annexin-V-FITC/PI Cytometric Analysis --- p.50 / Chapter 2.2.2.3 --- Determination of Mitochondrial Membrane Potential in cells --- p.51 / Chapter 2.2.2.4 --- Determination of Hydrogen Peroxide (H2O2) Release in cells --- p.52 / Chapter 2.2.2.5 --- Measurement of doxorubicin accumulation in cells --- p.53 / Chapter 2.2.2.6 --- Determination of P-glycoprotein expression level in cells --- p.54 / Chapter 2.2.2.7 --- Determination of mitochondrial depolarization and swellingin isolated mitochondria --- p.54 / Chapter 2.2.3 --- Methods involved in DNA sequencing of MDRl promoter --- p.56 / Chapter 2.2.3.1 --- DNA extraction --- p.56 / Chapter 2.2.3.2 --- DNA and Gel Band Purification --- p.56 / Chapter 2.2.3.3 --- Assessment of DNA amount --- p.57 / Chapter 2.2.3.4 --- Polymerase Chain Reaction --- p.57 / Chapter 2.2.3.5 --- Agarose Gel Electrophoresis --- p.59 / Chapter 2.2.3.6 --- Preparation for DNA sequencing --- p.59 / Chapter 2.2.4 --- Western Blotting Analysis --- p.61 / Chapter 2.2.4.1 --- Preparation of Proteins from Cells --- p.61 / Chapter 2.2.4.2 --- Preparation of proteins from isolated mitochondria --- p.63 / Chapter 2.2.4.3 --- Protein analysis with Western analysis --- p.63 / Chapter 2.2.5 --- Confocal laser scanning microscopy (Confocal microscopy) --- p.66 / Chapter 2.2.5.1 --- Analysis with confocal microscopy --- p.66 / Chapter 2.2.5.2 --- Determination of mitochondrial changes in cells by confocal microscopy --- p.66 / Chapter 2.2.5.3 --- Determination of lysosomal rupture in cells by confocal microscopy --- p.67 / Chapter 2.2.6 --- Mitochondrial isolation --- p.68 / Chapter Chapter 3 --- Results 一 Resistance Mechanisms in RHepG2 cells --- p.69 / Chapter 3.1 --- Resistance of RHepG2 cells towards various chemical agents --- p.70 / Chapter 3.1.1 --- RHepG2 cells are resistant to doxorubicin --- p.70 / Chapter 3.1.2 --- RHepG2 cells are resistant to taxol --- p.72 / Chapter 3.1.3 --- RHepG2 cells are resistant to valinomycin --- p.74 / Chapter 3.2 --- Resistance mechanism in RHepG2 cells --- p.76 / Chapter 3.2.1 --- Reduced doxorubicin accumulation is observed in RHepG2 cells --- p.76 / Chapter 3.2.2 --- More P-glycoproteins on the cell surface was observed in RHepG2 cells --- p.80 / Chapter 3.2.3 --- Inhibition of P-glycoprotein activity increased doxorubicin accumulation in RHepG2 cells --- p.82 / Chapter 3.2.4 --- HepG2 and RHepG2 cells contain the same P-glycoprotein promoter region --- p.86 / Chapter 3.2.5 --- RHepG2 over-expressed Bcl-2 --- p.91 / Chapter 3.2.6 --- HepG2 and RHepG2 cells had the same level of Bax protein --- p.93 / Chapter Chapter 4 --- Results - Effects of Polyphyllin D in HepG2 and RHepG2 cells --- p.95 / Chapter 4.1 --- Cytotoxicity of Polyphyllin D in HepG2 and RHepG2 cells --- p.96 / Chapter 4.1.1 --- Polyphyllin D exhibited cytotoxic effect in both HepG2 and RHepG2 cells --- p.96 / Chapter 4.2 --- Apoptotic mechanism caused by Polyphyllin D in HepG2 and RHepG2 cells --- p.93 / Chapter 4.2.1 --- Polyphyllin D caused apoptosis in HepG2 and RHepG2 cells --- p.98 / Chapter 4.2.2 --- Polyphyllin D did not activate caspase8 --- p.103 / Chapter 4.2.3 --- Polyphyllin D did not concentrate on the plasma membrane of cells --- p.106 / Chapter 4.2.4 --- Polyphyllin D did not change Bcl-2 level in HepG2 and RHepG2 cells --- p.109 / Chapter 4.2.5 --- Polyphyllin D treatment enhanced Bax protein expression in both HepG2 and RHepG2 cells --- p.111 / Chapter 4.2.6 --- Polyphyllin D caused cytochrome c and AIF release in HepG2 and RHepG2 cells --- p.113 / Chapter 4.2.7 --- Polyphyllin D induced mitochondrial membrane depolarization in HepG2 and RHepG2 cells --- p.118 / Chapter 4.2.8 --- Polyphyllin D caused mitochondrial swelling in HepG2 and clustering of mitochondriain RHepG2 cells --- p.122 / Chapter 4.2.9 --- "Polyphyllin D caused H202 release in HepG2 and RHepG2 cells, and the cytotoxic effects of Polyphyllin D could be reduced by NAC" --- p.127 / Chapter 4.2.10 --- Polyphyllin D caused permeabilization of lysosomes --- p.132 / Chapter 4.3 --- Site of action of Polyphyllin D in cells --- p.135 / Chapter 4.3.1 --- Purity of isolated mitochondria --- p.135 / Chapter 4.3.2 --- Polyphyllin D caused cytochrome c release from the HepG2 and RHepG2 isolated mitochondria --- p.137 / Chapter 4.3.3 --- Polyphyllin D induced mitochondrial depolarization in HepG2 and RHepG2 isolated mitochondria --- p.139 / Chapter 4.3.4 --- Polyphyllin D caused mitochondrial swelling in HepG2 and RHepG2 isolated mitochondria --- p.142 / Chapter 4.4 --- Resistance reversal effects of Polyphyllin D in RHepG2 cells --- p.144 / Chapter 4.4.1 --- Polyphyllin D increased doxorubicin accumulation in RHepG2 cells --- p.144 / Chapter 4.4.2 --- P-glycoprotein expression was not down-regulated after Polyphyllin D treatment --- p.146 / Chapter 4.4.3 --- Co-treatment of doxorubicin with Polyphyllin D had enhanced cytotoxic effect --- p.148 / Chapter Chapter 5 --- Discussion - Resistance mechanisms in RHepG2 cells --- p.150 / Chapter 5.1 --- Resistance of RHepG2 cells towards various chemical reagents --- p.151 / Chapter 5.2 --- Resistance mechanisms in RHepG2 cells --- p.154 / Chapter Chapter 6 --- Discussion - Effects of Polyphyllin D in HepG2and RHepG2 cells --- p.159 / Chapter 6.1 --- Cytotoxicity of Polyphyllin D in HepG2 and RHepG2 cells --- p.160 / Chapter 6.2 --- Apoptotic mechanisms caused by Polyphyllin D in HepG2 and RHepG2 cells --- p.162 / Chapter 6.3 --- Site of action of Polyphyllin D in HepG2 andRHepG2 cells --- p.172 / Chapter 6.4 --- Resistance reversal effects of Polyphyllin D in RHepG2 cells --- p.175 / Chapter Chapter 7 --- Future Perspectives --- p.177 / Chapter Chapter 8 --- Conclusion --- p.179 / References
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Characterization of two ras-superfamily members, RhoC and Rab14, in hepatocellular carcinoma (HCC).January 2004 (has links)
Lau Yee Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 147-157). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / List of Figures --- p.viii / List of Tables --- p.xi / Contents --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.1 --- Background of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.2 --- Etiology of HCC --- p.2 / Chapter 1.1.3 --- Relationship between HCC and HBV --- p.3 / Chapter 1.1.4 --- Differential gene expression under induction of HBx protein by microarray analysis --- p.5 / Chapter 1.1.5 --- Confirmation of candidate genes --- p.6 / Chapter 1.2 --- Ras-Oncogene --- p.8 / Chapter 1.2.1 --- Ras superfamily --- p.8 / Chapter 1.2.1.1 --- Rho family --- p.9 / Chapter 1.2.1.2 --- Rab family --- p.10 / Chapter 1.2.2 --- Functional mechanism of small GTPase --- p.11 / Chapter 1.2.3 --- Possible functions of Rho and Rab family members --- p.14 / Chapter 1.3 --- RhoC --- p.16 / Chapter 1.3.1 --- The genomic and protein structures of RhoC --- p.16 / Chapter 1.3.2 --- Relationship between RhoC and tumours --- p.19 / Chapter 1.4 --- Rabl4 --- p.20 / Chapter 1.4.1 --- The genomic and protein structures of Rabl4 --- p.20 / Chapter 1.4.2 --- Relationship between Rabl4 and tumours --- p.23 / Chapter 1.5 --- Aims of study --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Cell lines --- p.25 / Chapter 2.1.2 --- Cell culture reagents --- p.26 / Chapter 2.1.3 --- Reagents for total RNA isolation --- p.29 / Chapter 2.1.4 --- Reagents for reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 2.1.5 --- Reagents and buffers for Western blot analysis --- p.31 / Chapter 2.1.6 --- Vectors for cloning --- p.39 / Chapter 2.1.7 --- Reagents for polymerase chain reaction (PCR) --- p.39 / Chapter 2.1.8 --- Restriction digestion reagents --- p.42 / Chapter 2.1.9 --- Reagents for agarose gel electrophoresis --- p.42 / Chapter 2.1.10 --- Ligation reagents --- p.44 / Chapter 2.1.11 --- Bacterial culture medium --- p.44 / Chapter 2.1.12 --- Dyes and reagents for fluorescent microscope --- p.46 / Chapter 2.1.13 --- Reagents for flow cytometry --- p.48 / Chapter 2.1.14 --- Detection of apoptosis --- p.48 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Identification of gene expression of candidate genes in HCC --- p.50 / Chapter 2.2.1.1 --- cDNA preparation --- p.50 / Chapter (1) --- Cell culture of HepG2 and WRL-68 cell lines --- p.50 / Chapter (2) --- Total RNA isolation --- p.50 / Chapter (3) --- First-strand cDNA synthesis --- p.51 / Chapter 2.2.1.2 --- RT-PCR of candidate genes --- p.52 / Chapter 2.2.1.3 --- Western blotting --- p.53 / Chapter (1) --- Cell culture --- p.53 / Chapter (2) --- Protein extraction --- p.53 / Chapter (3) --- Quantification of proteins --- p.53 / Chapter (4) --- Detection of RhoC and Rabl4 protein by western blot analysis --- p.54 / Chapter (5) --- Western blotting luminol detection --- p.56 / Chapter 2.2.2 --- Cloning protocol --- p.57 / Chapter 2.2.2.1 --- Amplification of RhoC and Rabl4 genes --- p.57 / Chapter 2.2.2.2 --- Purification of PCR product --- p.58 / Chapter 2.2.2.3 --- Restriction enzymes digestion --- p.53 / Chapter 2.2.2.4 --- Insert/vector ligation --- p.59 / Chapter 2.2.2.5 --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.60 / Chapter 2.2.2.6 --- Transformation of ligation product into chemically competent bacterial cells --- p.61 / Chapter 2.2.2.7 --- Small-scale preparation of bacterial plasmid DNA --- p.61 / Chapter 2.2.2.8 --- Screening for recombinant clones --- p.62 / Chapter 2.2.2.9 --- DNA sequencing of cloned plasmid DNA --- p.63 / Chapter 2.2.2.10 --- Midi-scale preparation of recombinant plasmid DNA --- p.64 / Chapter 2.2.3 --- Visualization of the subcellular localization patterns --- p.66 / Chapter 2.2.3.1 --- Cell culture of AML12 and HepG2 cell lines --- p.66 / Chapter 2.2.3.2 --- Transfection of GFP fusion constructs into cells --- p.66 / Chapter 2.2.3.3 --- DAPI staining --- p.67 / Chapter 2.2.3.4 --- ER-Tracker´ёØ Blue-White DPX staining --- p.68 / Chapter 2.2.3.5 --- Subcellular localization study using Epi-fluorescence microscopy --- p.68 / Chapter 2.2.4 --- Analysis of cell cycle --- p.69 / Chapter 2.2.4.1 --- Transfection of GFP vectors / GFP-tagged proteins into cells --- p.69 / Chapter 2.2.4.2 --- Analysis of cell cycle by flow cytometry --- p.69 / Chapter 2.2.5 --- Detection of apoptosis --- p.70 / Chapter 2.2.5.1 --- Transfection --- p.70 / Chapter 2.2.5.2 --- Detection of DNA fragmentation --- p.70 / Chapter 2.2.6 --- Reorganization of Actin cytoskeleton by RhoC --- p.71 / Chapter 2.2.6.1 --- Transfection of GFP vectors/GFP-tagged proteins into cells --- p.71 / Chapter 2.2.6.2 --- Rhodamine phalloidin (RP) staining --- p.71 / Chapter 2.2.6.3 --- Epi-fluorescence microscopy --- p.72 / Chapter 2.2.7 --- Analysis of cell invasion under induction of RhoC --- p.72 / Chapter 2.2.7.1 --- "Sub-cloning of human RhoC gene into a mammalian expression vector, pHM6" --- p.72 / Chapter 2.2.7.2 --- Transfection of pHM6-RhoC --- p.73 / Chapter 2.2.7.3 --- Cell invasion assay --- p.73 / Chapter 2.2.8 --- Analysis of downstream effectors in RhoC-mediated pathway --- p.75 / Chapter 2.2.8.1 --- RT-PCR --- p.75 / Chapter 2.2.8.2 --- Western blotting --- p.75 / Chapter 2.2.9 --- Analysis of role of Rabl4 in membrane trafficking --- p.76 / Chapter 2.2.9.1 --- Cloning and transfection --- p.76 / Chapter 2.2.9.2 --- Alexa 594 transferrin conjugate staining --- p.76 / Chapter 2.2.9.3 --- Epi-fluorescence microscopy --- p.77 / Chapter 2.2.10 --- Statistics --- p.77 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Expression of RhoC and Rabl4 in hepatoma cells --- p.78 / Chapter 3.1.1 --- RT-PCR --- p.78 / Chapter 3.1.2 --- Western blotting --- p.81 / Chapter 3.2 --- Subcellular localization of RhoC and Rab 14 --- p.85 / Chapter 3.3 --- Characterization of RhoC --- p.93 / Chapter 3.3.1 --- Cell cycle analysis --- p.93 / Chapter 3.3.2 --- Apoptosis --- p.95 / Chapter 3.3.3 --- Actin cytoskeleton reorganization --- p.97 / Chapter 3.3.4 --- Cell invasion ability --- p.99 / Chapter 3.3.5 --- Downstream effectors of RhoC in cytoskeletal reorganization --- p.102 / Chapter 3.4 --- Characterization of Rabl4 --- p.107 / Chapter 3.4.1 --- Cell cycle analysis --- p.107 / Chapter 3.4.2 --- Apoptosis --- p.109 / Chapter 3.4.3 --- Roles in intracellular transportation --- p.111 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Strong expression of RhoC and Rabl4 in hepatoma cells --- p.117 / Chapter 4.2 --- Subcellular localization of RhoC and Rabl4 --- p.119 / Chapter 4.3 --- The effects of RhoC in normal liver cells --- p.122 / Chapter 4.3.1 --- Cell cycle progression by RhoC through regulating of G1 to S phase transition --- p.122 / Chapter 4.3.2 --- RhoC shows no apoptotic effect in normal liver cell systems --- p.123 / Chapter 4.3.3 --- Formation of actin filaments and stress fibers --- p.124 / Chapter 4.3.4 --- Induction of cell invasion in RhoC-expressing cells --- p.125 / Chapter 4.3.5 --- Downstream effectors in signaling pathway of RhoC in actin filment reorganization and cell invasion --- p.126 / Chapter 4.4 --- The effects of Rabl4 in normal liver cells --- p.132 / Chapter 4.4.1 --- Cell proliferation effects of Rabl4 by increasing percentage of cells in S phase for DNA synthesis --- p.132 / Chapter 4.4.2 --- Rabl4 has no apoptotic effects --- p.133 / Chapter 4.4.3 --- Roles of Rabl4 in vesicular transport --- p.134 / Chapter 4.5 --- Conclusion --- p.138 / Chapter 4.6 --- Future prospects --- p.140 / Appendix --- p.143 / References --- p.147
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