A comparative analysis of survivin distribution in young, aged and transformed human cells, and its influence on genomic integrity under hypoxia.

Genome instability increases with age and is a hallmark of cancer, unsurprisingly, therefore, age is the primary risk factor in cancer (1). The aim of this project is to compare the genomic integrity (nuclear and mitochondria (mt)) of young versus old fibroblasts and transformed cells that differentially express the inhibitor of apoptosis protein, survivin. Owing to the presence of cell cycle related elements in its promoter region, in normal cells survivin is usually only expressed in G2 and M phase, however, its expression is upregulated under hypoxia, a state that has relevance to both ageing and to cancer. Literature suggests that as cells age, survivin switches from being predominantly nuclear to mainly cytoplasmic (2), and that this redistribution enables cells to tolerate stress, giving them a survival advantage. In cancer cells survivin becomes additionally localised to the mitochondria. We have extensive data to show that in cancer cells mitochondrial survivin inhibits mitophagy, the specialist from of autophagy that eliminates damaged mitochondria: consequently the cells bear extensive mtDNA lesions and switch from a metabolic dependency on Oxphos to glycolysis, known as the "Warburg transition". In this project young, old and transformed cells will be grown under hypoxic conditions, and normoxic conditions, survivin distribution and relative abundance recorded, and suppressed using the commercially available inhibitor, YM155, and the survivin-specific miRNA, miR203. Cell proliferation and viability will be assessed using colony counting and FACS analysis, and a resazurin-resarufin assay used to determine respiratory capacity. Cell health and mitochondrial integrity will be assessed by live imaging with fluorescently labelled mitotracker, and nuclear DNA repair monitored using anti-H2AX foci in fixed preparations. To determine genomic integrity directly, RNA will isolated from young (passage 2-4) and aged (passage 35) fibroblasts and cancer cells, reverse transcribed and the integrity of the nuclear and mitochondrial DNA monitored using PCR reactions designed to generate short DNA (200bp) and long (7kb) fragments from each genome. Reactions from cells harbouring nuclear and mtDNA lesions will yield fewer long PCR products, but will have many short fragments, while cells with undamaged DNA will deliver plenty of both products. Preliminary data from my lab suggest that survivin overexpression in cancer cells inhibits mitophagy and causes an accumulation of mt lesions (SPW, A. Townley, in preparation), but whether this holds true under hypoxic conditions, or is affected by ageing, has not been addressed. Data from this project will enable us to determine whether survivin expression increases cellular tolerance to nuclear and mtDNA damage, facilitates nuclear DNA repair, and/or accelerates mitochondrial clearance, whether these responses differ with age and how survivin modulates these responses, and whether any intervention to survivin function in these pathways could have therapeutic potential.