Single-Cell Analysis of p16INK4a and p21WAF1 Expression Suggests Distinct Mechanisms of Senescence in Normal Human and Li-Fraumeni Syndrome Fibroblasts
Single-Cell Analysis of p16INK4a and p21WAF1 Expression Suggests Distinct Mechanisms of Senescence in Normal Human and Li-Fraumeni Syndrome Fibroblasts
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DOI：
10.1002/jcp.22002
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发表时间：
2010-04-01
影响因子：
5.6
通讯作者：
Murray, David
中科院分区：
文献类型：
--
作者：
Mirzayans, Razmik;Andrais, Bonnie;Murray, David
本文采用单细胞观察方法，探讨p16（INK4A）和p21(WAF1)（以下简称p16和p21）在人[正常、失调性毛细血管扩张（AT）和Li-Fraumeni综合征（LFS）]成纤维细胞增殖性衰老和电离辐射诱导的加速衰老中的作用。所有菌株的培养在传代后期进入复制衰老状态，从生长抑制、获得扁平和扩大的细胞形态以及衰老相关的β -半乳糖苷酶阳性染色可以看出这一点。此外，这些菌株的增殖早期培养物在电离辐射下表现出加速衰老的反应。免疫荧光显微镜显示p16在正常和AT成纤维细胞株中的异质表达，与体外培养年龄无关，大多数细胞均未检测到p16的水平。重要的是，在p53精通（正常和AT）的成纤维细胞中，复制性衰老和电离辐射引发的加速衰老伴随着p21的持续核积累，但与p16的表达无关。另一方面，在p53缺陷（LFS）成纤维细胞中，复制性衰老和电离辐射触发的加速衰老与pi6的表达密切相关，而与p21的表达无关。此外，LFS成纤维细胞的衰老与包含多倍体的基因组不稳定性有关。我们的研究结果与p16作为衰老的备用调节因子的模型相一致，在缺乏野生型p53活性的情况下优先触发这种反应。这些结果也支持了一种可能性，即pi6的一种肿瘤抑制功能可能与基因组不稳定性有关，从而阻止多倍体巨细胞产生恶性后代。j .细胞。中国生物医学工程学报，2009,31(2):557 - 567。(C) 2009 Wiley-Liss, Inc。
Herein we used single-cell observation methods to gain insight into the roles of p16(INK4A) and p21(WAF1) (hereafter p16 and p21) in replicative senescence and ionizing radiation-induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li-Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence-associated beta-galactosidase. In addition, proliferating early-passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53-proficient (normal and AT) fibroblasts. In p53-deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation-triggered accelerated senescence strongly correlated with expression of p I 6 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild-type p53 activity. The possibility that one of the tumor-suppressor functions of p I 6 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 57-67, 2010. (C) 2009 Wiley-Liss, Inc.