Independent induction of senescence by p16INK4a and p21CIP1 in spontaneously immortalized human fibroblasts.
Independent induction of senescence by p16INK4a and p21CIP1 in spontaneously immortalized human fibroblasts.
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发表时间：
1998-02
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通讯作者：
M. Vogt;C. Haggblom;J. Yeargin;T. Christiansen-Weber;M. Haas
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作者：
M. Vogt;C. Haggblom;J. Yeargin;T. Christiansen-Weber;M. Haas
在这项工作中，我们解决了是否可以在永生的非致瘤性人类成纤维细胞中诱导复制衰老的问题。本研究中使用的永生成纤维细胞来自两名 Li-Fraumeni (LF) 患者，他们的种系中携带一种野生型和一种突变型 p53 等位基因。两个永生系都丢失了 wtp53 等位基因，并且不表达可检测到的 p16INK4a 蛋白，尽管它们携带 p16INK4a 基因。与永生的人类成纤维细胞相比，衰老的人类成纤维细胞的 DNA 中 5-甲基胞嘧啶含量较低。这一观察结果表明，去甲基化剂可能会恢复永生表型并诱导永生细胞系的复制性衰老。将两个 LF 系的细胞暴露于去甲基化剂 5-aza-2'-脱氧胞苷。 6 天内，所有细胞生长停滞，并显示出衰老细胞增大且平坦的形态特征、脂褐素颗粒的积累以及 pH6 下衰老相关的 β-半乳糖苷酶活性，这都是衰老的生物标志物。 5-aza-2'-脱氧胞苷处理的细胞的免疫印迹显示 p16INK4a 蛋白的表达大大增加，但 p21CIP1 的表达没有检测到变化，p21CIP1 是一种已知在衰老的正常人成纤维细胞中强烈表达的基因。在另外两个实验系列中，两个 LF 系的细胞被编码 p16INK4a 或 p21CIP1 的逆转录病毒构建体感染。每个转导的基因都诱导衰老而不影响其他内源基因的表达。结果表明，永生 LF 成纤维细胞的衰老诱导可以通过不同的途径发生：（a）通过诱导 p16INK4a 表达的去甲基化依赖性途径； (b) 通过涉及 p21CIP1 表达的去甲基化独立途径。 p16INK4a 和 p21CIP1 诱导衰老的情况在两个人永生成纤维细胞系中发生的情况相同，但其端粒长度和端粒酶活性不同。
In this work, we address the question of whether replicative senescence can be induced in immortal nontumorigenic human fibroblasts. The immortal fibroblasts used in this study were derived from two Li-Fraumeni (LF) patients who carry in their germ line one wild-type and one mutant p53 allele. Both immortal lines have lost the wtp53 allele and express no detectable p16INK4a protein, although they carry the p16INK4a gene. In contrast to immortal human fibroblasts, senescent human fibroblasts have a low content of 5-methyl-cytosine in their DNA. This observation suggested the possibility that a demethylating agent could revert the immortal phenotype and induce replicative senescence in the immortal cell lines. Cells of the two LF lines were exposed to the demethylating agent 5-aza-2'-deoxycytidine. Within 6 days, all cells were growth arrested and showed the enlarged and flat morphology characteristic of senescent cells, an accumulation of lipofuscin granules and senescence-associated beta-galactosidase activity at pH6, both biomarkers for senescence. Immunoblots of 5-aza-2'-deoxycytidine-treated cells showed a greatly increased expression of p16INK4a protein but no detectable change in the expression of p21CIP1, a gene known to be strongly expressed in senescent normal human fibroblasts. In two other experimental series, cells of the two LF lines were infected with retroviral constructs encoding either p16INK4a or p21CIP1. Each of the transduced genes induced senescence without affecting the expression of the other endogenous gene. The results show that induction of senescence in immortal LF fibroblasts can occur by different pathways: (a) by demethylation-dependent pathways that induce the expression of p16INK4a; and (b) by demethylation-independent pathways involving the expression of p21CIP1. The induction of senescence by p16INK4a and p21CIP1 occurred equally in the two human immortal fibroblast lines, which differed in the length of their telomeres and the activity of their telomerase.