Reconstitution of active telomerase in primary human foreskin fibroblasts: effects on proliferative characteristics and response to ionizing radiation
Reconstitution of active telomerase in primary human foreskin fibroblasts: effects on proliferative characteristics and response to ionizing radiation
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DOI：
10.1080/09553000410001692735
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发表时间：
2004-05-01
影响因子：
2.6
通讯作者：
Linskens, MHK
中科院分区：
文献类型：
--
作者：
Kampinga, HH;Van Waarde-Verhagen, MAWH;Linskens, MHK
目的：端粒缩短被认为是引发衰老的原因，并且由于大多数原代细胞不表达活性端粒酶，因此端粒酶活性的重新激活被认为是一种安全且非转化的永生化细胞的方式。然而，为了研究辐射反应，目前还不清楚通过端粒酶再活化而永生化的细胞是否以与其亲本原代细胞相似的方式表现。材料和方法：用端粒酶的人催化亚单位逆转录酶（hTERT）转染原代人包皮成纤维细胞，并表征其生长特性和对DNA损伤的反应。人hTERT的单独表达足以使人包皮成纤维细胞永生化。随着培养时间的推移，永生化细胞的平均端粒长度几乎增加了一倍，克隆群体几乎不再含有有丝分裂后的成纤维细胞。与亲本原代细胞相比，高达300个群体倍增，在克隆形成辐射敏感性、DNA双链断裂修复、辐射诱导的p53和p21（WAF-1，CIP-1）表达增加以及G1/S和G2/M细胞周期检查点方面没有观察到改变。此外，有丝分裂原诱导的有丝分裂阻滞的成纤维细胞仍然是可能的hTERT-永生化clones.Conclusions：永生化成纤维细胞的活性端粒酶重建似乎是一个很好的，可靠的方式来产生一个大的来源的细胞与辐射损伤反应类似的原代细胞。
Purpose : Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non-transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear whether cells immortalized by telomerase reactivation behave in a similar manner as their parental primary cells.Materials and methods : Primary human foreskin fibroblasts were transfected with the human catalytic subunit of telomerase, the reverse transcriptase (hTERT), and their growth characteristics and response to DNA damage were characterized.Results : The sole expression of the human hTERT was sufficient to immortalize the human foreskin fibroblasts. With time in culture, the immortalized cells almost doubled their average telomeric length and the clonal population contained almost no post-mitotic fibroblasts anymore. Up to 300 population doublings, no alterations compared with the parental primary cells were seen in terms of clonogenic radiosensitivity, DNA double-strand break repair, radiation-induced increases in p53 and p21(WAF-1,CIP-1) expression, and the G1/S and G2/M cell cycle checkpoints. Moreover, mitogen-induced mitotic arrest of fibroblasts was still possible in the hTERT-immortalized clones.Conclusions : Immortalizing fibroblasts by reconstitution of active telomerase seems a good, reliable manner to generate a large source of cells with a radiation damage response similar to the primary cells.