In situ analysis of changes in telomere size during replicative aging and cell transformation.
In situ analysis of changes in telomere size during replicative aging and cell transformation.
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DOI：
10.1083/jcb.134.1.1
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发表时间：
1996-07
期刊：
影响因子：
--
通讯作者：
Harley C
中科院分区：
文献类型：
--
作者：
Henderson S;Allsopp R;Spector D;Wang SS;Harley C
Southern分析表明，在人类体细胞的复制老化过程中，端粒逐渐缩短。本研究采用荧光原位杂交技术（FISH）在单细胞水平检测端粒缩短。间期人二倍体成纤维细胞（HDFs）的FISH和共聚焦显微镜表明，端粒分布在整个细胞核的染色体间的异质性的大小。在不断增加的群体倍增水平下对HDF的分析显示，端粒信号的斑点大小、强度和可检测性逐渐增加。从年轻和年老的HDFs制备的中期染色体的FISH显示1、9、15和Y染色体上端粒的检测频率的异质性。早期和晚期传代细胞的检测频率的染色体间分布相似。中期染色体端粒检出率随年龄增长而降低。这些观察结果表明，端粒缩短在正常人类体细胞中以相似的速率。T-抗原转化的HDFs危机附近含有端粒信号低相比，nontransformed HDFs。一个大的细胞内的端粒长度的异质性，检测到在两个端粒酶阴性细胞系相比，正常的体细胞和端粒酶阳性293细胞系。许多端粒酶阴性的永生细胞的端粒信号强于年轻的HDFs，这表明端粒酶阴性的永生细胞的端粒长度调节机制不同。这些研究提供了端粒长度的染色体间异质性的原位证明。此外，FISH是在单细胞水平上检测端粒大小变化的可靠且灵敏的方法。
Telomeres have been shown to gradually shorten during replicative aging in human somatic cells by Southern analysis. This study examines telomere shortening at the single cell level by fluorescence in situ hybridization (FISH). FISH and confocal microscopy of interphase human diploid fibroblasts (HDFs) demonstrate that telomeres are distributed throughout the nucleus with an interchromosomal heterogeneity in size. Analysis of HDFs at increasing population doubling levels shows a gradual increase in spot size, intensity, and detectability of telomeric signal. FISH of metaphase chromosomes prepared from young and old HDFs shows a heterogeneity in detection frequency for telomeres on chromosomes 1, 9, 15, and Y. The interchromosomal distribution of detection frequencies was similar for cells at early and late passage. The telomeric detection frequency for metaphase chromosomes also decreased with age. These observations suggest that telomeres shorten at similar rates in normal human somatic cels. T-antigen transformed HDFs near crisis contained telomere signals that were low compared to nontransformed HDFs. A large intracellular heterogeneity in telomere lengths was detected in two telomerase-negative cell lines compared to normal somatic cells and the telomerase-positive 293 cell line. Many telomerase-negative immortal cells had telomeric signals stronger than those in young HDFs, suggesting a different mechanism for telomere length regulation in telomerase-negative immortal cells. These studies provide an in situ demonstration of interchromosomal heterogeneity in telomere lengths. Furthermore, FISH is a reliable and sensitive method for detecting changes in telomere size at the single cell level.