No description available.

Nck is as an adaptor protein previously implicated in actin cytoskeleton reorganization and by our laboratory, in translation and cell response to stress. Our primary objective was to determine the expression levels of Nck isotypes (Nck-1 and Nck-2) during cancer progression. We have performed western blot analysis of the Nck isotypes expression levels profile in various human cancer cell lines, at different stages of progression. Our data show significantly higher expression levels of Nck-2 protein in metastatic melanomas compared to non-metastatic melanomas and normal melanocytes. Using semi-quantitative RT-PCR, we demonstrated that this increase in Nck-2 expression can be also seen at the transcriptional level. The Ras/RAF/MEK/ERK pathway is often spontaneously activated in melanomas causing hyperactivation of ERK. By downregulating the expression of Nck-2 using siRNA, we have established a strong correlation between increased expression levels of Nck-2 and activated ERK. Furthermore, we have demonstrated the involvement of Nck-2 in cell proliferation and adhesion in metastatic melanomas, revealing that Nck-2 acts as a new player in this disease.

Leung Chun-on, Paul. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 149-167). / Content Page / Acknowledgments --- p.7 / Chapter / Chapter Chapter1 --- Introduction --- p.8 / Chapter Chapter2 --- Literature Review --- p.13 / Chapter 2.1 --- Anatomy of the cervix --- p.13 / Chapter 2.2 --- Classification --- p.14 / Chapter 2.2.1 --- Cervical intraepithelial neoplasia (CIN) --- p.14 / Chapter 2.2.2 --- Cervical cancer --- p.17 / Chapter 2.2.3 --- Incidence and screening --- p.21 / Chapter 2.2.4 --- Etiology / Chapter 2.2.4.1 --- Sexual and reproductive factors --- p.23 / Chapter 2.2.4.2 --- Smoking as a risk factor --- p.23 / Chapter 2.2.4.3 --- Male partner contribution --- p.24 / Chapter 2.2.4.4 --- Human papillomaviruses and cervical cancer --- p.24 / Chapter 2.2.4.5 --- Oral contraceptive pills --- p.27 / Chapter 2.2.4.6 --- Oncogenes and tumour suppresser genes --- p.28 / Chapter 2.2.4.7 --- Oncogenes and cervical cancer --- p.35 / Chapter 2.3 --- Immunohistochemical technique in cancer study / Chapter 2.3.1 --- Principle of immunostaining --- p.39 / Chapter 2.3.2 --- Fixation --- p.40 / Chapter 2.3.3 --- Section preparation --- p.41 / Chapter 2.3.4 --- The choice of antibodies --- p.41 / Chapter 2.3.5 --- Enzyme labels --- p.42 / Chapter 2.3.6 --- Blocking endogenous enzymes --- p.43 / Chapter 2.3.7 --- Blocking background staining --- p.43 / Chapter 2.3.8 --- Dilution preparation --- p.44 / Chapter 2.3.9 --- The Avidin-Biotin technique --- p.44 / Chapter 2.3.10 --- Control --- p.47 / Chapter 2.3.11 --- Antigen retrieval --- p.47 / Chapter 2.3.12 --- Cell counting and scoring --- p.49 / Chapter 2.4 --- The application of Polymerase Chain Reaction Single-Strand Conformation Polymorphism(PCR-SSCP) in cancer study --- p.52 / Chapter Chapter3 --- Materials and Methods --- p.56 / Chapter 3.1 --- Materials --- p.56 / Chapter 3.2 --- Methods --- p.61 / Chapter 3.2.1 --- Specimens collection --- p.61 / Chapter 3.2.2 --- Antibodies preparation --- p.63 / Chapter 3.2.3 --- Immunohistochemical staining and antigen retrieval procedures --- p.63 / Chapter 3.2.4 --- Cell counting and scoring --- p.68 / Chapter 3.2.5 --- PCR-SSCP analysis for myc gene mutation --- p.70 / Chapter 3.2.5.1 --- DNA extraction --- p.70 / Chapter 3.2.5.2 --- PCR --- p.72 / Chapter 3.2.5.3 --- Preparing the single strand DNA --- p.73 / Chapter 3.2.5.4 --- Electrophoresis --- p.73 / Chapter 3.2.5.5 --- Gel drying and scanning --- p.77 / Chapter 3.2.6 --- Statistical analysis --- p.77 / Chapter Chapter 4 --- Result --- p.78 / Chapter Chapter 5 --- Discussion --- p.126 / Chapter Chapter 6 --- Conclusions --- p.144 / Reference --- p.148

Tsang Sup-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 176-185). / Abstracts in English and Chinese. / Acknowledgments --- p.1 / Abstract --- p.2 / 摘要 --- p.4 / List of Abbreviation --- p.6 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Isolation and activation of mas oncogene --- p.11 / Chapter 1.2 --- Amino acid sequence of mas oncogene --- p.14 / Chapter 1.3 --- Expression of mas oncogene --- p.18 / Chapter 1.4 --- Possible physiological role of mas oncogene --- p.20 / Chapter 1.5 --- Gene related to mas family --- p.23 / Chapter 1.6 --- Aims of study --- p.26 / Chapter Chapter 2 --- Over-expression of mas oncogene / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.2 --- Materials and Methods --- p.29 / Chapter 2.2.1 --- Materials --- p.30 / Chapter 2.2.1.1 --- Chemicals --- p.30 / Chapter 2.2.1.2 --- Enzyme --- p.30 / Chapter 2.2.1.3 --- DNA Purification Kit --- p.31 / Chapter 2.2.1.4 --- Others --- p.31 / Chapter 2.2.2 --- Methods --- p.31 / Chapter 2.2.2.1 --- Strategy of construct preparation --- p.31 / Chapter 2.2.2.2 --- "Preparation of linearized vector, pFRSV" --- p.32 / Chapter 2.2.2.2.1 --- Cloning of vectors --- p.32 / Chapter 2.2.2.2.2 --- Restriction enzyme digestion and DNA dephosphorylation --- p.34 / Chapter 2.2.2.2.3 --- DNA purification by agarose gel electro-elution --- p.34 / Chapter 2.2.2.3 --- Preparation of pFRSV/mas construct --- p.35 / Chapter 2.2.2.3.1 --- PCR amplification --- p.35 / Chapter 2.2.2.3.2 --- Restriction enzyme digestion --- p.36 / Chapter 2.2.2.4 --- Ligation and analysis --- p.37 / Chapter 2.2.2.5 --- Purification of DNA by cesium chloride --- p.38 / Chapter 2.2.2.5.1 --- Large-scale bacterial culturing --- p.38 / Chapter 2.2.2.5.2 --- Ethanol precipitation --- p.39 / Chapter 2.2.2.5.3 --- Cesium chloride purification --- p.39 / Chapter 2.2.2.5.4 --- Removal of DNA dye by dialysis and ethanol precipitation --- p.40 / Chapter 2.2.2.6 --- Transfection by electroporation --- p.41 / Chapter 2.2.2.7 --- Screening for the stably transfected cells --- p.42 / Chapter 2.2.2.8 --- RT-PCR analysis of the mas transfectant --- p.43 / Chapter 2.2.2.8.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.43 / Chapter 2.2.2.8.2 --- Reverse transcription of the total RNA into cDNA --- p.44 / Chapter 2.2.2.8.3 --- Analysis of the transfected mas expression by PCR --- p.44 / Chapter 2.2.2.8.4 --- Analysis of the transfected DHFR expression by PCR --- p.45 / Chapter 2.2.2.8.5 --- Analysis of endogenous GAPDH expression by PCR --- p.46 / Chapter 2.2.2.9 --- Amplification of mas transgene by using methotrexate --- p.47 / Chapter 2.2.2.9.1 --- Amplification by low dosage MTX treatment --- p.47 / Chapter 2.2.2.9.2 --- Amplification by high dosage MTX treatment --- p.49 / Chapter 2.2.2.10 --- Southern blot analysis --- p.50 / Chapter 2.2.2.10.1 --- Preparation of DIG-labelled mas probe --- p.51 / Chapter 2.2.2.10.2 --- Preparation of DIG-labelled DHFR probe --- p.51 / Chapter 2.2.2.10.3 --- Preparation of DIG-labelled GAPDH probe --- p.52 / Chapter 2.2.2.10.4 --- Isolation of Genomic DNA from the mas transfectants by DNAzol® Reagent / Chapter 2.2.2.10.5 --- Enzymatic restriction of genomic DNA and Gel electrophoresis --- p.54 / Chapter 2.2.2.10.6 --- DNA transferring to positive charged Nylon membrane --- p.54 / Chapter 2.2.2.10.7 --- Pre-hybridization and hybridization --- p.56 / Chapter 2.2.2.10.8 --- Post-hybridization washing and blocking --- p.56 / Chapter 2.2.2.10.9 --- Detection --- p.57 / Chapter 2.2.2.11 --- Northern blot analysis --- p.57 / Chapter 2.2.2.11.1 --- Preparation of the agarose gel containing formaldehyde --- p.58 / Chapter 2.2.2.11.2 --- Preparation of the RNA sample --- p.58 / Chapter 2.2.2.11.3 --- Gel electrophoresis and transferring --- p.59 / Chapter 2.2.2.11.5 --- Pre-hybridization and hybridization --- p.60 / Chapter 2.2.2.11.4 --- Post-hybridization washing and blocking --- p.60 / Chapter 2.2.2.11.6 --- Detection --- p.61 / Chapter 2.2.2.11.7 --- Stripping and rehybridization --- p.61 / Chapter 2.3 --- Results --- p.62 / Chapter 2.3.1 --- RT-PCR analysis of gene expression in the stably transfectant --- p.62 / Chapter 2.3.2 --- Morphology of the mas trasnfectant --- p.64 / Chapter 2.3.3 --- Determination of mas gene copy number by Southern blot analysis in the mas transfectants --- p.66 / Chapter 2.3.4 --- Northern blot analysis of the transcriptional level of mas transcriptsin mas transfectants --- p.76 / Chapter 2.4 --- Discussion --- p.87 / Chapter Chapter 3 --- In vivo study of physiological effect of over-expression of mas / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Materials and Methods --- p.93 / Chapter 3.2.1 --- Materials --- p.93 / Chapter 3.2.2 --- Methods --- p.93 / Chapter 3.2.2.1 --- Cell culture --- p.93 / Chapter 3.2.2.2 --- Subcutaneous injection of nude mice --- p.94 / Chapter 3.2.2.3 --- Isolation of the total RNA from the tumor tissues --- p.95 / Chapter 3.2.2.4 --- Northern blot analysis --- p.96 / Chapter 3.3 --- Results --- p.96 / Chapter 3.3.1 --- Tumorgenicity assay of mas oncogene in nude mice --- p.96 / Chapter 3.3.2 --- Northern blot analysis of mas expression in the tumor tissues --- p.103 / Chapter 3.4 --- Discussion --- p.109 / Chapter Chapter 4 --- Fluorescent differential display analysis of mas transfectants / Chapter 4.1 --- Introduction --- p.111 / Chapter 4.2 --- Materials and Methods --- p.112 / Chapter 4.2.1 --- Materials --- p.112 / Chapter 4.2.1.1 --- Chemicals --- p.112 / Chapter 4.2.1.2 --- Enzyme --- p.113 / Chapter 4.2.1.3 --- Kits --- p.113 / Chapter 4.2.1.4 --- Others --- p.114 / Chapter 4.2.2 --- Methods --- p.114 / Chapter 4.2.2.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.114 / Chapter 4.2.2.2 --- DNase I treatment --- p.115 / Chapter 4.2.2.3 --- Reverse transcription (RT) and non-fluorescent PCR --- p.116 / Chapter 4.2.2.4 --- Reverse transcription and fluorescent differential display-PCR --- p.118 / Chapter 4.2.2.5 --- High resolution fluorescent differential display (Fluoro DD) gel --- p.118 / Chapter 4.2.2.6 --- Gel band excision of differentially expressed cDNA fragments --- p.120 / Chapter 4.2.2.7 --- Gel band reamplification --- p.120 / Chapter 4.2.2.8 --- Subcloning of reamplified cDNA fragments --- p.121 / Chapter 4.2.2.9 --- Purification of plasmid DNA from recombinant clones for reverse dot blot analysis --- p.122 / Chapter 4.2.2.10 --- Reverse dot blot analysis --- p.123 / Chapter 4.2.2.10.1 --- Preparation of cDNA dot blot --- p.123 / Chapter 4.2.2.10.2 --- Preparation of DIG-labeled cDNA library probes --- p.124 / Chapter 4.2.2.10.3 --- Hybridization --- p.126 / Chapter 4.2.2.11 --- Northern blot analysis --- p.127 / Chapter 4.3 --- Results --- p.128 / Chapter 4.3.1 --- Fluorescent differential display (FluoroDD) --- p.128 / Chapter 4.3.2 --- Reverse dot blot analysis --- p.135 / Chapter 4.3.3 --- DNA sequencing analysis of the clones --- p.141 / Chapter 4.3.4 --- Confirmation of differential display pattern of the subclones by Northern blot analysis --- p.160 / Chapter 4.4 --- Discussion --- p.166 / Chapter Chapter 5 --- General Discussion / Chapter 5.1 --- General model for mos-induced tumor formation --- p.169 / Chapter 5.2 --- Future aspect --- p.174 / References --- p.176 / Appendix I Buffer composition --- p.186 / Appendix II Sequences of fluoroDD TMR-Anchored primers and arbitrary primers --- p.190

Human papillomavirus (HPV) infections play a crucial role in human carcinogenesis. Greater than 96% of all cervical carcinomas are positive for high-risk HPV infections; especially types 16 and 18. High-risk HPV onco-proteins, E6 and E7, are consistently expressed in such cancers and function by inactivating p53 and pRb tumor suppressors, respectively. The presence of high-risk HPVs is also correlated with anogenital cancers. In this study, we examined the effect of high-risk HPV type 16 E6 and E7 oncoproteins in two normal human colorectal epithelial cell lines, NCE1 and NCE5. We report that the expression of E6/E7 proteins, alone, induced cellular transformation of both cell lines; consequently, NCE1-E6/E7 and NCE5-E6/E7 form colonies in soft agar with respect to their wild type cells. This is accompanied by cell cycle deregulation, as is demonstrated by the over-expression of cyclin dependant kinases (cdks) and their respective cyclins. Furthermore, we demonstrate that E6/E7 oncoprotein transduction induces migration of colorectal epithelial cells. More still, well analyzed Id gene expression, a family member of the helix-loop-helix (HLH) transcription factors involved in the regulation of cell invasion and metastasis of human cancer cells. In parallel, using tissue microarray analysis we found that the four members of the Id protein family are correlated with the presence of HPV type 16 and 18 in human colon cancer tissues. Our data suggests that high-risk HPV infections are sufficient to induce cellular transformation of normal human colorectal cells, in vitro. Furthermore, the correlation with the Id family of proteins may present a novel set of markers associated with HPV induced colorectal carcinogenesis. Our results may suggest a new approach to detect and prevent colorectal cancer.

Thesis (Ph. D.)--Cornell University, January, 1989. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Biochemistry. Bibliography: leaves 83-87. Also available online.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [74]-91).