p53 Tumor Suppressor Pathway

真核生物基因组不断面临外源和内源诱变剂的破坏威胁。因此，哺乳动物细胞进化出了一个复杂的防御网络来维持基因组的稳定。P53处于这些防御途径的十字路口。端粒保护蛋白1(POT1)是护色素复合体的单链DNA结合成分，对端粒特异的t-环结构的形成起着至关重要的作用，是端粒长度的调节因子。与端粒相关因子-2(TRF2)在这些过程中也发挥作用的观察一致，我们报道了POT1和TRF2在保护端粒和调节细胞衰老和凋亡方面的合作。POT1的siRNA敲除导致端粒单链突起(3‘突起)的丢失、染色体的不稳定、乳腺癌细胞的凋亡和正常成纤维细胞的衰老。POT1在物理上与TRF2相互作用，并保护其免受显性负性TRF2诱导的端粒功能障碍。我们还发现，人类POT1(V5)的C末端截短变体保护端粒并防止细胞衰老，与全长POT1一样有效，但在机制上不同：(A)全长POT1，而不是v5，通过维持3‘悬臂发挥作用；(B)p53参与v5基因敲除引起的衰老；以及(C)v5仅在一小部分端粒上发挥作用。基于这些结果，我们认为人类端粒受到功能上不同的POT1变体的协同调控。端粒保护变异体v5在染色体稳定的错配修复缺陷肿瘤中优先表达，提示v5在人类癌症染色体稳定中的作用。我们正在继续研究控制端粒磨损的POT1或P53亚型调控的P53介导的复制性衰老。正常人类细胞有限的增殖能力导致复制性细胞衰老，这是体内肿瘤进展的关键障碍。我们已经证明，人类P53亚型(β133P53和P53β)在P53介导的复制性衰老的内源性调节机制中发挥作用。在正常人成纤维细胞中，p53β的诱导和delta133p53的降低与复制性衰老有关，而与癌基因诱导的衰老无关。复制衰老的成纤维细胞还表达高水平的miR-34a，这是一种由p53诱导的microRNA，其反义抑制延缓了复制衰老的开始。短干扰RNA(SiRNA)介导的内源性delta133p53基因敲除导致细胞衰老，这归因于对p21waf1和其他p53转录靶基因的调控。在过表达实验中，虽然p53beta与全长p53协同促进细胞衰老，但delta133p53抑制miR-34a的表达并延长细胞复制寿命，提供了这种microRNA与p53异构体介导的衰老调节的功能联系。在具有衰老表型的结肠腺瘤中，观察到与衰老相关的P53亚型表达的特征(即P53β升高和Delta133P53降低)。在结肠癌中发现的delta13p53增加和p53β亚型表达降低可能是从腺瘤向癌发展过程中逃离衰老屏障的信号。

The eukaryote genome constantly faces the threat of damage from exogenous and endogenous mutagens. Mammalian cells, therefore, have evolved an intricate network of defenses to maintain genomic stability. p53 is at the crossroads of these defense pathways. Protection of Telomeres 1(POT1)is a single stranded DNA binding component of the shelterin complex, which is essential to the formation of a telomere-specific t-loop structure and functions as a regulator of telomere length. Consistent with observations that Telomere-Related Factor-2 (TRF2) also functions in these processes, we reported that POT1 and TRF2 cooperated to protect telomeres and regulate cellular senescence and apoptosis. The siRNA knockdown of POT1 resulted in the loss of telomeric single-stranded overhangs (3' overhangs), chromosomal instability, apoptosis in breast cancer cells, and cellular senescence in normal human fibroblasts. POT1 physically interacted with TRF2 and protected against the dominant-negative TRF2-induced telomere dysfunction. We also found that a C-terminally truncated variant of human POT1 (v5) protected telomeres and prevented cellular senescence as effeciently as the full-length POT1, but in mechanistically different manners: (a) the full-length POT1, but not v5, functions through the maintenance of 3' overhangs; (b) p53 is dispensible to v5 knockdown-induced senescence; and (c) v5 functions at only a fraction of telomeres. Based on these results, we propose that human telomeres are cooperatively regulated by the functionally distinct POT1 variants. The telomere-protective variant v5 is preferentially expressed in mismatch repair-deficient tumors, which have stable chromosomes, suggesting the role of v5 in chromosome stability in human cancers. We are continuing to study p53-mediated replicative senescence modulated by POT1 or p53 isoforms that govern telomere attrition. The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier in tumour progression in vivo. We have shown that human p53 isoforms (delta 133p53 and p53beta) function in an endogenous regulatory mechanism for p53-mediated replicative senescence. Induced p53beta and diminished delta133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA, the antisense inhibition of which delayed the onset of replicative senescence. The short interfering RNA (siRNA)- mediated knockdown of endogenous delta133p53 induced cellular senescence, which was attributed to the regulation of p21waf1 and other p53 transcriptional target genes. In overexpression experiments, whereas p53beta cooperated with full-length p53 to accelarate cellular senescence, delta133p53 repressed miR-34a expression and extended the cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (that is, elevated p53beta and reduced delta133p53) was observed in vivo in colon adenomas with senescent phenotypes. The increased delta13p53 and decreased p53beta isoform expression found in colon carcinoma may signal an escape from the senescence barrier during the progression from adenoma to carcinoma.