Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts
Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts
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DOI：
10.1073/pnas.93.24.13742
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发表时间：
1996-11-26
影响因子：
11.1
通讯作者：
Barrett, JC
中科院分区：
文献类型：
--
作者：
Alcorta, DA;Xiong, Y;Barrett, JC
人二倍体成纤维细胞（HDF）可以在培养基中生长有限数量的群体倍增时间，然后停止增殖并进入称为复制性衰老的生长停滞状态。在这些细胞中表达的视网膜母细胞瘤基因产物Rb是低磷酸化的。为了确定衰老人成纤维细胞维持低磷酸化Rb的可能机制，我们检测了衰老HDF中Rb激酶CDK4和CDK6以及细胞周期蛋白依赖性激酶抑制剂p21和p16的表达水平和相互作用，细胞p21蛋白表达在最后两到三代中显著增加，此时大多数细胞失去其生长潜力并接近衰老，p21在衰老HDFs中表达下降，p16 mRNA和细胞蛋白表达逐渐升高，衰老HDFs中p16蛋白水平比早代细胞高近10倍。在衰老的HDF中，p16被证明与CDK4和CDK6复合。对衰老细胞提取物中p21和p16的免疫耗竭分析表明，p16是CDK4和CDK6激酶的主要CDK抑制剂，对来自衰老HDF的放射性标记提取物中的CDK4和CDK6及其相关蛋白的免疫沉淀显示没有其他CDK抑制剂。基于这些结果，我们提出衰老是一个多步骤的过程，需要p21和p16的表达。p16上调是衰老中生长停滞的终末阶段的关键事件，这可以解释为什么p16而不是p21通常在永生细胞和人类肿瘤中突变。
Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and Interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs, Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs, During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly IO-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 Is the major CDK inhibitor for both CDK4 and CDK6 kinases, Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors, Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16, p16 up-regulation is a key event in the terminal stages of growth arrest In senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.