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Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer
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,...
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| Format: | Thesis |
| Language: | English |
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Division of Clinical Haematology
2016
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| _version_ | 1867613755242708992 |
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| access_status_str | Open Access |
| author | Godsmark, Grant Kenneth |
| author2 | Mowla, Shaheen |
| author_browse | Godsmark, Grant Kenneth Mowla, Shaheen |
| author_facet | Mowla, Shaheen Godsmark, Grant Kenneth |
| author_sort | Godsmark, Grant Kenneth |
| collection | Thesis |
| description | 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. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/20405 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:41:11.342Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2016 |
| publishDateRange | 2016 |
| publishDateSort | 2016 |
| publisher | Division of Clinical Haematology |
| publisherStr | Division of Clinical Haematology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/20405 Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer Godsmark, Grant Kenneth Mowla, Shaheen Medicine 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. 2016-07-18T12:42:04Z 2016-07-18T12:42:04Z 2016 Master Thesis Masters MSc (Med) http://hdl.handle.net/11427/20405 eng application/pdf Division of Clinical Haematology Faculty of Health Sciences University of Cape Town |
| spellingShingle | Medicine Godsmark, Grant Kenneth Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer |
| thesis_degree_str | Master's |
| title | Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer |
| title_full | Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer |
| title_fullStr | Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer |
| title_full_unstemmed | Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer |
| title_short | Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer |
| title_sort | investigation of the role of the dna modifying enzyme activation induced cytidine deaminase aid in cancer |
| topic | Medicine |
| url | http://hdl.handle.net/11427/20405 |
| work_keys_str_mv | AT godsmarkgrantkenneth investigationoftheroleofthednamodifyingenzymeactivationinducedcytidinedeaminaseaidincancer |