Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer

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dc.contributor.advisorMowla, Shaheenen_ZA
dc.contributor.authorGodsmark, Grant Kennethen_ZA
dc.date.accessioned2016-07-18T12:42:04Z
dc.date.available2016-07-18T12:42:04Z
dc.date.issued2016en_ZA
dc.description.abstractThe DNA-editing enzyme, Activation-Induced cytidine Deaminase (AID) is essential for antibody diversification and plays an important role in immunity. AID is specifically expressed in activated B-cells and mutates targeted DNA sites, diversifying the antibody repertoire. Due to its mutagenic nature, AID expression is tightly regulated, however, its overexpression has been associated with translocation of the c-MYC oncogene, a characteristic of the B-cell derived cancer, Burkitt's lymphoma (BL). Although currently uncharacterised, AID overexpression has also been implicated in non-lymphoid cancers including prostate, liver and colon. The function of AID in the oncogenic process is not well defined and therefore this project is aimed at using cell culture models to study the function of AID in cancer. AID mRNA and protein expression levels were determined in five cell lines of B-cell origin, and 16 epithelial cell lines (colon, prostate, head and neck and oesophageal). While AID expression was easily detected in the B-cell derived cell lines, no significant expression of both AID mRNA and protein expression was found in all the epithelial derived cell lines. The B lymphoblastoid cell line, L1439A, which is derived from a healthy donor, and harboured relatively low AID expression, was originally selected for ectopic expression of AID. Transfection of this cell line using conventional methods and lipid and polymer based transfection reagents was not successful, and therefore, nucleofection was used, which caused successful uptake of the AID-expressing plasmids as well as corresponding controls. However, this method was too harsh for the L1439A cell line as it did not survive post-nucleofection. Based on this result, the BL cell line Ramos, which expresses relatively low AID compared to the other BL cell lines used in the screen, was selected as an alternative. Plasmids constitutively expressing AID, as well as their corresponding empty vector, were successfully introduced into these cells using an established nucleofection protocol. While cells containing the empty vectors could be selected and expanded in culture, cells overexpressing AID underwent apoptosis approximately three days post-transfection. This is likely due to the highly mutagenic nature of the enzyme. As an alternative approach to studying the function of AID in cancer, a knockdown approach was taken, using siRNA.en_ZA
dc.identifier.apacitationGodsmark, G. K. (2016). <i>Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Clinical Haematology. Retrieved from http://hdl.handle.net/11427/20405en_ZA
dc.identifier.chicagocitationGodsmark, Grant Kenneth. <i>"Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Clinical Haematology, 2016. http://hdl.handle.net/11427/20405en_ZA
dc.identifier.citationGodsmark, G. 2016. Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Godsmark, Grant Kenneth AB - The DNA-editing enzyme, Activation-Induced cytidine Deaminase (AID) is essential for antibody diversification and plays an important role in immunity. AID is specifically expressed in activated B-cells and mutates targeted DNA sites, diversifying the antibody repertoire. Due to its mutagenic nature, AID expression is tightly regulated, however, its overexpression has been associated with translocation of the c-MYC oncogene, a characteristic of the B-cell derived cancer, Burkitt's lymphoma (BL). Although currently uncharacterised, AID overexpression has also been implicated in non-lymphoid cancers including prostate, liver and colon. The function of AID in the oncogenic process is not well defined and therefore this project is aimed at using cell culture models to study the function of AID in cancer. AID mRNA and protein expression levels were determined in five cell lines of B-cell origin, and 16 epithelial cell lines (colon, prostate, head and neck and oesophageal). While AID expression was easily detected in the B-cell derived cell lines, no significant expression of both AID mRNA and protein expression was found in all the epithelial derived cell lines. The B lymphoblastoid cell line, L1439A, which is derived from a healthy donor, and harboured relatively low AID expression, was originally selected for ectopic expression of AID. Transfection of this cell line using conventional methods and lipid and polymer based transfection reagents was not successful, and therefore, nucleofection was used, which caused successful uptake of the AID-expressing plasmids as well as corresponding controls. However, this method was too harsh for the L1439A cell line as it did not survive post-nucleofection. Based on this result, the BL cell line Ramos, which expresses relatively low AID compared to the other BL cell lines used in the screen, was selected as an alternative. Plasmids constitutively expressing AID, as well as their corresponding empty vector, were successfully introduced into these cells using an established nucleofection protocol. While cells containing the empty vectors could be selected and expanded in culture, cells overexpressing AID underwent apoptosis approximately three days post-transfection. This is likely due to the highly mutagenic nature of the enzyme. As an alternative approach to studying the function of AID in cancer, a knockdown approach was taken, using siRNA. DA - 2016 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2016 T1 - Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer TI - Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer UR - http://hdl.handle.net/11427/20405 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/20405
dc.identifier.vancouvercitationGodsmark GK. Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Clinical Haematology, 2016 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/20405en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Clinical Haematologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMedicineen_ZA
dc.titleInvestigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in canceren_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMSc (Med)en_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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