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

基因组不稳定性随着年龄的增长而增加，是癌症的标志，因此，年龄是癌症的主要风险因素，这并不奇怪。这个项目的目的是比较年轻与老年成纤维细胞和转化细胞的基因组完整性（细胞核和线粒体（mt）），这些细胞差异表达凋亡抑制蛋白生存素。由于在其启动子区存在细胞周期相关元件，在正常细胞中，存活素通常仅在G2和M期表达，然而，其表达在缺氧下上调，缺氧状态与衰老和癌症都相关。文献表明，随着细胞的衰老，生存素从主要的细胞核转变为主要的细胞质（2），这种重新分布使细胞能够耐受压力，使它们具有生存优势。在癌细胞中，存活素变得另外定位于线粒体。我们有大量的数据表明，在癌细胞中，线粒体生存素抑制线粒体自噬，自噬的专家形式，消除受损的线粒体：因此，细胞承担广泛的mtDNA损伤，并从对Oxphos的代谢依赖转换为糖酵解，称为“瓦尔堡转变”。在这个项目中，年轻的，年老的和转化的细胞将在低氧条件下生长，和常氧条件下，记录存活素分布和相对丰度，并使用市售的抑制剂YM 155和存活素特异性miRNA miR 203抑制。将使用菌落计数和FACS分析评估细胞增殖和活力，并使用刃天青-刃芦灵测定法确定呼吸能力。将通过使用荧光标记的mitotracker进行实时成像来评估细胞健康和线粒体完整性，并使用固定制备物中的抗H2 AX焦点监测核DNA修复。为了直接确定基因组完整性，将从年轻（第2-4代）和老化（第35代）成纤维细胞和癌细胞中分离RNA，进行逆转录，并使用设计用于从每个基因组生成短DNA（200 bp）和长（7 kb）片段的PCR反应监测核和线粒体DNA的完整性。来自具有核和mtDNA损伤的细胞的反应将产生较少的长PCR产物，但将具有许多短片段，而具有未损伤DNA的细胞将提供大量的两种产物。来自我实验室的初步数据表明，存活素在癌细胞中的过表达抑制了线粒体自噬并导致线粒体损伤的积累（SPW，A. Townley，在准备中），但这是否适用于缺氧条件下，或受老化的影响，还没有得到解决。来自该项目的数据将使我们能够确定生存素表达是否增加细胞对核和mtDNA损伤的耐受性，促进核DNA修复和/或加速线粒体清除，这些反应是否随年龄而不同，生存素如何调节这些反应，以及对生存素在这些途径中的功能的任何干预是否具有治疗潜力。

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.