Mechanism of in-vitro aging

描述（由申请人提供）：大多数人类细胞在分化后不久停止端粒酶的产生，随后经历染色体末端（称为端粒）的逐渐缩短。在培养的原代人细胞中也观察到在体内正常衰老过程中看到的端粒丢失和其他一些变化，这些细胞在大约50个细胞周期后停止分裂（称为复制性衰老）。端粒酶缺陷细胞的模式真核生物酿酒酵母（芽殖酵母）也表现出端粒缩短和衰老。衰老的确切原因尚不清楚，但有证据表明切外切酶切除缩短的端粒会导致染色体不稳定。这种不稳定性的特征是染色体丢失和缺失事件，以及导致双中心融合和其他畸变的重排。目的1：一个新的，可调节的端粒酶表达系统将用于测试提出的细胞衰老机制。致命染色体重排模型将通过询问在衰老晚期停止生长的细胞是否可以通过端粒酶的重新激活来拯救来进行严格的测试。实验还将分析(a)稳定双着丝点功能的着丝点缺陷，(b) DNA损伤检查点反应，(c)端粒的核酸外切酶切除。染色体畸变频率和端粒长度的抢救细胞将被评估。目的2：将检测正常、检查点缺陷和切除缺陷的单倍体细胞在“死亡边缘”通过与未受损细胞交配而获救的能力，形成具有短染色体和良好拷贝补充的二倍体。将监测稳定的二倍体的畸变频率。目的3：确定化学促氧化剂和抗氧化剂对衰老的影响。在抗氧化DNA损伤所需基因失活的细胞中，存活和端粒完整性也将被监测。目的4：将采用一种新的方法来表征衰老旁路机制，包括鉴定体外细胞衰老的高复制抑制因子。染色体缩短和许多其他代谢变化发生在正常的人类衰老过程中，也发生在培养的细胞中。提出的实验将确定遗传和环境因素，调节细胞衰老，也可能在体内发挥作用。在衰老过程中，大多数人体组织细胞中的染色体逐渐缩短，随之而来的DNA不稳定与癌症和与年龄相关的疾病的增加有关。拟议的研究将加强我们对由于染色体缩短而发生的事件和影响缩短速度的因素的理解。

DESCRIPTION (provided by applicant): Most human cells halt production of the enzyme telomerase shortly after differentiation and subsequently experience progressive shortening of chromosome ends, called telomeres. Telomere loss and several other changes seen during normal aging in vivo are also observed in primary human cells grown in culture, which stop dividing after approximately 50 cell cycles (called replicative senescence). Telomerase-deficient cells of the model eukaryote Saccharomyces cerevisiae (budding yeast) also exhibit telomere-shortening and senescence. The precise cause of senescence is unknown, but evidence suggests that exonuclease resection of the shortened telomeres leads to chromosome instability. This instability is characterized by chromosome loss and deletion events, as well as rearrangements leading to dicentric fusions and other aberrations. Aim 1: A new, regulatable telomerase expression system will be used to test proposed mechanisms of cell senescence. The lethal chromosome rearrangement model will be critically tested by asking if cells that stop growing in late senescence can be rescued by reactivation of telomerase. Experiments will also analyze mutants defective in (a) kinetochore functions that stabilize dicentrics, (b) DNA damage checkpoint responses, and (c) exonuclease-resection of telomeres. Frequencies of chromosome aberrations and telomere lengths in the rescued cells will be assessed. Aim 2: The ability of normal, checkpoint-, and resection-defective haploid cells at "the brink of death" to be rescued by mating to undamaged cells, forming diploids with shortened chromosomes complemented by good copies, will be assayed. Frequencies of aberrations in the stabilized diploids will be monitored. Aim 3: The impact of well-characterized chemical pro-oxidants and antioxidants on senescence will be determined. Survival and telomere integrity will also be monitored in cells in which genes required for resistance to oxidative DNA damage have been inactivated. Aim 4: A new approach to characterization of senescence bypass mechanisms will be employed that involves identification of high copy suppressors of in vitro cell aging. Chromosome shortening and many other metabolic changes occur during both normal human aging and also in cells grown in culture. The proposed experiments will identify genetic and environmental factors that modulate cellular aging that may also function in vivo. Chromosomes become progressively shortened in cells of most human tissues during aging, with subsequent DNA instability linked to increases in cancer and age-associated diseases. The proposed research will enhance our understanding of events that occur as a consequence of shortened chromosomes and factors that affect the rate of shortening.