Catalytically active human telomerase mutants with allele-specific biological properties
Catalytically active human telomerase mutants with allele-specific biological properties
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DOI：
10.1016/s0014-4827(03)00217-9
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发表时间：
2003-08-15
影响因子：
3.7
通讯作者：
Blackburn, EH
中科院分区：
文献类型：
--
作者：
Kim, M;Xu, LF;Blackburn, EH
人端粒酶催化亚基 hTERT 的表达可延长人原代成纤维细胞的寿命。据报道，这种寿命的延长通常伴随着端粒的净延长，这导致了这样的假设：端粒的延长导致了寿命的延长。在这里，我们发现 hTERT+C 和 hTERT-FlagC（在 hTERT C 末端添加了 10 个额外残基或 FLAG 表位的突变端粒酶蛋白）赋予 IMR90 人原代肺成纤维细胞显着但有限的寿命延长。然而，当细胞继续生长超过100次群体倍增超过其正常衰老点时，大量端粒长度继续侵蚀至比对照端粒酶阴性细胞衰老时所见的长度短得多。由三种不同癌基因转化的 hTERT+C 永生化 IMR90 细胞的表达。同样，在危机中，大量端粒变得比对照细胞短得多。其他 hTERT 突变体的构建和分析类似。与之前报道的其他保守的 GQ 结构域突变体和 C 端 HA 标记的 hTERT 一样，具有酶活性的 hTERT-125A+T16A 未能延长寿命。另一种 GQ 结构域突变体 hTERT-E79A 在细胞生长效应方面与前端野生型 hTERT 无法区分，但没有净端粒延长。这些结果进一步揭示了 hTERT 等位基因特异性表型，可解开端粒酶活性、净端粒延长和寿命延长。 (C) 2003 年爱思唯尔科学（美国）。版权所有。
Expression of the catalytic subunit of human telomerase, hTERT, extends human primary fibroblast life span. Such life span extension has generally been reported to be accompanied by net telomere lengthening, which led to the hypothesis that it is the telomere lengthening that causes the life span extension. Here we show that hTERT+C and hTERT-FlagC, mutant telomerase proteins with either 10 additional residues or a FLAG epitope added to the hTERT C-terminus, confer significant but limited life span extension to IMR90 human primary lung fibroblasts. However, as the cells continue to grow for >100 population doublings past their normal senescence point, bulk telomere length continues to erode to length much shorter than those seen at the senescence of control telomerase-negative cells. Expression of hTERT+C immortalized IMR90 cells transformed by three different oncogenes. Again, bulk telomeres became much shorter than those of the control cells at crisis. Additional hTERT mutants were constructed and analyzed similarly. Enzymatically active hTERT-125A+T16A, like other previously reported conserved GQ domain mutants and C-terminally HA-tagged hTERT, failed to extend life span. Another GQ domain mutant, hTERT-E79A, was indistinguishable front wild-type hTERT in its cell growth effects, but there was no net telomere lengthening. These results uncover further hTERT allele-specific phenotypes that uncouple telomerase activity, net telomere lengthening and life span extension. (C) 2003 Elsevier Science (USA). All rights reserved.