A characterisation of genes involved in apoptosis resistance

Davis, Tanja (2013-03)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Apoptosis represents a finely orchestrated and highly conserved natural form of cell death. It exhibits unique morphological and biochemical characteristics which culminate in the controlled dismantling of a cell from within followed by its discreet removal by phagocytic cells. Apoptosis is vital for the preservation of cell and tissue homeostasis but also performs several defensive and protective functions. Owing to its importance, apoptosis is highly regulated and a large number of proteins have been shown to mediate and safeguard the process. Furthermore, deregulated or altered levels of apoptosis can have severe pathological consequences; indeed, apoptosis has been shown to play a central role in several diseases, including neurological and autoimmune diseases as well as a variety of cancers. Consequently, the search for apoptotic-based therapies has received much attention and of vital importance to this quest is the characterisation of the specific mediators of apoptosis and their regulation as well as the identification of novel genes or proteins that can have a regulatory effect on apoptosis. It is thus the aim of this study to assist in this characterisation and also to identify novel candidate genes potentially involved in apoptosis. In a previously performed pilot study, three novel candidate genes potentially involved in apoptosis were identified by performing promoter-trap mutagenesis experiments. These genes were lipoic acid synthetase (LIAS), cyclophilin A (CYPA) and ribosomal protein L9 (RPL9). Since the methodology for this pilot study involved the use of functionally haploid cells, it was aimed in this study to verify these results in a diploid mouse cell line. Candidate gene knockdown was achieved by means of RNA interference and apoptosis assays were performed. A potential role for LIAS and CYPA in apoptosis was successfully verified in this study; however this could not be achieved for RPL9 and the gene was thus excluded from further study. In addition, nucleotide sequences isolated during the promoter-trap mutagenesis experiments in the pilot study were also investigated in order to identify additional novel candidate genes involved in apoptosis. By performing nucleotide BLAST searches, two potential candidate genes were identified, namely AHNAK nucleoprotein (AHNAK) and serum amyloid A-like 1 (SAAL1). Further bioinformatic analyses were performed with the four candidate genes in order to ascertain possible associations with apoptosis or cancer. Lastly, to further characterise the four candidate genes, the relative gene expression was investigated by means of quantitative PCR in two cancer and control cell lines. The results revealed significant differential expression for the majority of genes in the cancer cell lines when compared to the control cell lines. In conclusion, this study identified and characterised four novel genes potentially involved in apoptosis. Results obtained during this study can aid in the complete characterisation and functional annotation of these genes. Potential ties to apoptosis and associations with cancer are discussed for all four candidate genes and the possibilities of therapeutic strategies for anticancer treatments involving these candidate genes are noted.

AFRIKAANSE OPSOMMING: Apoptose verteenwoordig ‘n fyn georganiseerde en hoogs gekonserveerde natuurlike vorm van seldood. Dit vertoon unieke morfologiese and biochemiese eienskappe wat uitloop in die beheerde afbreek van ‘n sel vanuit die binnekant waarna dit onopsigtelik deur fagositiese selle verywder word. Apoptose is uiters belangrik vir die bewaring van sel en weefsel homeostase, maar dit vervul ook menigde afwerende and beskermde funksies. Vanweë sy noodsaaklikheid is apoptose hoogs gereguleer and ‘n groot aantal proteïene is al aangewys as bemiddelaars en beskermers van die proses. Verder, wangereguleerde en veranderde vlakke van apoptose kan ernstige patalogiese nagevolge hê; inderdaad, ‘n sentrale rol vir apoptose in verskeie siektes is al bevestig, insluitend neurologiese en outo-immuun siektes asook ‘n verskeidenheid van kankers. As gevolg hiervan ontvang die soektogte vir apoptose-gebaseerde terapieë vele aandag en uiters noodsaaklik vir hierdie soektogte is die karakterisering van die spesifieke bemiddelaars van apoptose en hul regulering asook die identifisering van nuwe gene of proteïene wat ‘n regulerende effek kan hê op apoptose. Dit is dus die doel van hierdie studie om by te dra tot hierdie karakterisering en ook om nuwe kandidaat gene wat moontlik betrokke kan wees in apoptose te identifiseer. In ‘n vorige loodsprojek is drie gene moontlik betrokke in apoptose geïdentifiseer deur middel van promoter-strik mutagenese eksperimente. Hierdie gene is lipoic acid synthetase (LIAS), cyclophilin A (CYPA) en ribosomal protein L9 (RPL9). Aangesien die metodiek in the loodsprojek gebruik gemaak het van funksionele haploïede selle, was dit die doel van hierdie studie om die resultate te bevestig in ‘n diploïede muis sellyn. Ribonukleïensuur (RNS) steuring is uitgevoer vir die uitklopping van die kandidaat gene en apoptose toetse is ook gedoen. Die bevestiging van ‘n moontlike rol vir LIAS en CYPA in apoptose was suksesvul in hierdie studie; alhoewel dit was nie bereikbaar vir RPL9 nie en hierdie geen is dus uitgesluit in verdere studies. Bykomend is nukleotied volgordes wat geïsoleer is tydens die promoter-strik mutagenese eksperimente in die loodsprojek ook nagesien om moontlike addisionele nuwe kandidaat gene te identifiseer wat moontlik betrokke kan wees by apoptose. Twee potensiële kandidaat gene, naamlik AHNAK nucleoprotein (AHNAK) en serum amyloid A-like 1 (SAAL1), was geïdentifiseer deur middel van nukleotied BLAST soektogte. Addisionele bioinformatiese analises is uitgevoer op die vier kandidaat gene om moontlike redes vir ‘n assosiasie met apoptose of kanker vas te stel. Laastens, om die kandidaat gene verder te karakteriseer, is daar ondersoek ingestel op die relatiewe geen uitdrukking van die kandidaat gene in twee kanker en twee normale sellyne. Die resultate het betekenisvolle differensiële regulering getoon vir meeste van die gene in die kanker sellyne in vergelyking met die normale sellyne. Ten slotte, vier kandidaat gene moontlik betrokke in apoptose is in die huidige studie geïdentifiseer en gekarakteriseer. Die resultate verwerf in hierdie studie kan moontlik bydra tot die volkome karakterisering en funksionele annotering van die kandidaat gene. Moontlike skakels met apoptose en assosiasies met kanker is bepreek vir die vier kandidaat gene en die moontlikheid van terapeutiese strategieë gebaseer rondom die kandidaat gene word ook genoem.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/80335
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