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，并且是癌症的一个标志，因此，年龄是癌症的主要危险因素(1)。该项目的目的是比较年轻和年老成纤维细胞的基因组完整性（核和线粒体（mt）），以及表达凋亡蛋白抑制剂survivin的转化细胞的差异。由于在其启动子区域存在细胞周期相关元件，在正常细胞中，survivin通常仅在G2和M期表达，然而，在缺氧状态下，其表达上调，这一状态与衰老和癌症有关。文献表明，随着细胞年龄的增长，生存素从主要是核转变为主要是细胞质(2)，这种重新分配使细胞能够承受压力，赋予它们生存优势。在癌细胞中，生存素被附加地定位于线粒体。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，自噬消除受损线粒体的专家：因此，细胞承受广泛的mtDNA损伤，并从对Oxphos的代谢依赖转变为糖酵解，称为“Warburg转变”。在这个项目中，年轻、年老和转化的细胞将在缺氧和常氧条件下生长，记录survivin的分布和相对丰度，并使用市售抑制剂YM155和survivin特异性miRNA miR203进行抑制。细胞增殖和活力将通过菌落计数和FACS分析进行评估，并使用resazurin-resarufin测定呼吸能力。细胞健康和线粒体完整性将通过荧光标记的有丝分裂跟踪器实时成像评估，并使用固定制剂中的抗h2ax病灶监测核DNA修复。为了直接确定基因组的完整性，将从年轻的（2-4代）和年老的（35代）成纤维细胞和癌细胞中分离RNA，进行逆转录，并使用PCR反应从每个基因组中生成短DNA （200bp）和长DNA （7kb）片段来监测核和线粒体DNA的完整性。含有核和mtDNA损伤的细胞的反应将产生较少的长PCR产物，但将产生许多短片段，而具有未受损DNA的细胞将产生大量的这两种产物。我实验室的初步数据表明，survivin在癌细胞中的过度表达会抑制有丝分裂并导致mt病变的积累（SPW， A. Townley, in preparation），但这是否在缺氧条件下成立，还是受到衰老的影响，还没有得到解决。该项目的数据将使我们能够确定survivin的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变化，survivin如何调节这些反应，以及在这些途径中对survivin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.