Telomerase upregulation is a postcrisis event during senescence bypass and immortalization of two Nijmegen breakage syndrome T cell cultures
Telomerase upregulation is a postcrisis event during senescence bypass and immortalization of two Nijmegen breakage syndrome T cell cultures
复制标题
DOI：
10.1111/j.1474-9726.2010.00550.x
复制
发表时间：
2010-04-01
期刊：
影响因子：
7.8
通讯作者：
Roos, Goran
中科院分区：
文献类型：
--
作者：
Degerman, Sofie;Siwicki, Jan Konrad;Roos, Goran
我们对永生化和端粒生物学的认识主要是基于在生长危机之前分析的遗传操作细胞和在生长危机之后分析的许多群体怀疑。一般认为，生长危机是端粒长度（TL）依赖性的，而摆脱危机与端粒酶逆转录酶（hTERT）基因表达增加、端粒酶活性上调和TL稳定有关。在这里，我们已经分析了人类T细胞的自发永生化的过程中，涉及衰老和端粒酶调控的途径。两种奈梅亨断裂综合征（NBS）T细胞培养物（S3 R和S4）显示逐渐的端粒磨损，直到一段时间的生长危机，然后是永生化细胞的生长。全基因组表达分析表明，前，早期后和后期危机细胞之间的差异。早期危机后细胞表现出对数生长曲线，非常短的端粒，并且值得注意的是，尽管几种负hTERT调节剂下调，hTERT或端粒酶活性没有增加（例如，G. FOS、JUND、SMAD3、RUNX2、TNF-α和TGF β-R2）。此后，cMYC mRNA的增加与hTERT表达的增加、端粒酶活性和短端粒的延长平行，表明hTERT转录的逐步激活涉及负调节因子的减少，然后是正调节因子的激活。基因表达分析表明，细胞通过下调ATM、CDKN 1B（p27）、CDKN 2D（p19）和ASF 1A基因表达，上调CDK 4、TWIST 1、TP 73 L（p63）和SYK基因表达，从而逃避生长危机。因此，端粒酶上调被认为是解偶联的逃避生长危机，而是在NBS T细胞培养物的永生化过程中的后期事件。
Our knowledge on immortalization and telomere biology is mainly based on genetically manipulated cells analyzed before and many population doublings post growth crisis. The general view is that growth crisis is telomere length (TL) dependent and that escape from crisis is coupled to increased expression of the telomerase reverse transcriptase (hTERT) gene, telomerase activity upregulation and TL stabilization. Here we have analyzed the process of spontaneous immortalization of human T cells, regarding pathways involved in senescence and telomerase regulation. Two Nijmegen breakage syndrome (NBS) T cell cultures (S3R and S4) showed gradual telomere attrition until a period of growth crisis followed by the outgrowth of immortalized cells. Whole genome expression analysis indicated differences between pre-, early post- and late postcrisis cells. Early postcrisis cells demonstrated a logarithmic growth curve, very short telomeres and, notably, no increase in hTERT or telomerase activity despite downregulation of several negative hTERT regulators (e. g. FOS, JUN D, SMAD3, RUNX2, TNF-alpha and TGF beta-R2). Thereafter, cMYC mRNA increased in parallel with increased hTERT expression, telomerase activity and elongation of short telomeres, indicating a step-wise activation of hTERT transcription involving reduction of negative regulators followed by activation of positive regulator(s). Gene expression analysis indicated that cells escaped growth crisis by deregulated DNA damage response and senescence controlling genes, including downregulation of ATM, CDKN1B (p27), CDKN2D (p19) and ASF1A and upregulation of CDK4, TWIST1, TP73L (p63) and SYK. Telomerase upregulation was thus found to be uncoupled to escape of growth crisis but rather a later event in the immortalization process of NBS T cell cultures.