Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses
Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses
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DOI：
10.1016/j.dnarep.2005.07.005
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发表时间：
2006-01-05
期刊：
影响因子：
3.8
通讯作者：
States, JC
中科院分区：
文献类型：
--
作者：
Porter, PC;Clark, DR;States, JC
永生化细胞的 p53 活性经常受到破坏，并且缺乏 p53 依赖性核苷酸切除修复 (NER)。我们假设端粒酶永生化不会改变 p53 介导的紫外线 (UV) 诱导的 DNA 损伤反应。 DNA 修复能力强的原代二倍体人成纤维细胞 (GM00024) 通过表达端粒酶的逆转录病毒转导而永生化。空逆转录病毒转导的细胞在几次倍增后衰老。端粒酶转导的 GM00024 细胞 (tGM24) 连续培养 6 个月（> 60 次倍增）。紫外线照射后的集落形成能力在 0 至 20 J/m(2) UVC 之间呈剂量依赖性（LD50 = 5.6 J/m(2)）。 p53 的积累与 p48(XPE/DDB2)、p21(CIP1/WAF1) 的诱导以及 p53-S15 的磷酸化具有紫外线剂量和时间依赖性。通过核浓缩测量UV剂量依赖性细胞凋亡。通过使用紫外线照射的放射性标记 DNA 探针进行电泳迁移率变动测定来监测，紫外线暴露诱导的紫外线损伤的 DNA 结合受到 p53 特异性 siRNA 转染的抑制。 p53 特异性 siRNA 转染还可以阻止 p48 的 UV 诱导，并提高通过集落形成能力测量的 UV 存活率。 DHFR 内环丁烷嘧啶二聚体 (CPD) 的链特异性 NER 在 tGM24 和 GM00024 细胞中是相同的。 CPD 从转录链中的去除在 6 小时内几乎完成，从非转录链中的 CPD 去除在 24 小时内完成了 73%。 tGM24 中紫外线诱导的 HPRT 突变与原代人成纤维细胞无法区分。这些广泛的发现表明，紫外线诱导的 DNA 损伤反应在端粒酶永生化细胞中保持完整。此外，端粒酶永生化提供了永久细胞系，用于测试选择性遗传操作对 NER 和诱变的直接影响，而无需繁殖以建立突变系。 (C) 2005 Elsevier B.V. 保留所有权利。
Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20 J/m(2) UVC (LD50 = 5.6 J/m(2)). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6 h and from the non-transcribed strand was 73% complete in 24 h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines. (C) 2005 Elsevier B.V. All rights reserved.