Id proteins in the regulation of mammalian cell cycle, growth and survival

Members of the Id family of helix-loop-helix protein function as antagonists of several classes of transcription factor. Numerous studies have documented how Id proteins affect mammalian cell growth and survival. However, it is not known how these effects are influenced by cell lineage and tumourigenic conversion. This research aimed to investigate Id proteins on growth and survival by gain- and loss-of-function analysis in a diverse range of mammalian cell lines representing both primary and immortalised/tumourigenic cell lines of different lineages. By using automated image analysis software (CellProfiler Analyst), cell cycle and apoptosis profiles of cell populations transfected with either Id protein or an Id protein antagonist including siRNA down-regulation of Id proteins have been investigated. Also, cell viability analysis with MTT assay was evaluated. The study also employed flow cytometric analysis using a mitochondrial membrane-potential marker of viable cells (DiOC6) and Propidium Iodide (PI) as a marker of non- viable necrotic cells. The data showed that Id3 is pro-apoptotic in normal and tumourigenic cells of embryonic and epithelial lineages. Over-expression of Id3 also arrested growth in non-tumourigenic embryonic fibroblasts and tumourigenic colon cancer cells. Analyses of loss-or gain-of- function mutants of Id proteins suggest that Id2 Ala5 may be loss-of-function mutant in non- tumourigenic cell lines of embryonic lineage. The study revealed that Id2 Asp5 may be a loss-of-function mutant in tumourigenic cell lines of both embryonic and epithelial lineages. Id2 HE also behaved as a null mutant in both non-tumourigenic and tumourigenic cells representing embryonic and epithelial lineages. Data obtained also implicate Id3 Asp5 as a gain-of-function mutant whilst Id3 Ala5 may represent a loss-of-function mutant in non- tumourigenic and tumourigenic cells of different lineages. Another important finding was that Id protein antagonist (aHLH) induced apoptosis and impaired cell growth irrespective of the cell lineage and tumourigenic conversion. The effects on cell growth and survival following down-regulation of Idl, Id2 and Id3 differ in embryonic, epithelial and endothelial cells. The study also suggests that cell death as a result of Id2 knock-down may occur via a different mechanism. The results reveal distinct functions of Id proteins in normal, tumourigenic and primary cells of different lineages, suggesting that Id protein functions are dependent on cell lineage and tumourigenic properties.EThOS - Electronic Theses Online ServiceGBUnited Kingdo