Department of Pathology, University of California, San Francisco, California, United States of America.
Department of Biochemistry and Biophysics, University of California, San Francisco, California, United States of America.
PLoS One. 2018 Dec 5;13(12):e0206525. doi: 10.1371/journal.pone.0206525. eCollection 2018.
The telomerase enzyme enables unlimited proliferation of most human cancer cells by elongating telomeres and preventing replicative senescence. Despite the critical importance of telomerase in cancer biology, challenges detecting telomerase activity and expression in individual cells have hindered the ability to study patterns of telomerase expression and function across heterogeneous cell populations. While sensitive assays to ascertain telomerase expression and function exist, these approaches have proven difficult to implement at the single cell level. Here, we validate in situ RNAscope detection of the telomerase TERT mRNA and couple this assay with our recently described TSQ1 method for in situ detection of telomere elongation. This approach enables detection of TERT expression, telomere length, and telomere elongation within individual cells of the population. Using this assay, we show that the heterogeneous telomere elongation observed across a HeLa cell population is in part driven by variable expression of the TERT gene. Furthermore, we show that the absence of detectable telomere elongation in some TERT-positive cells is the result of inhibition by the telomeric shelterin complex. This combined assay provides a new approach for understanding the integrated expression, function, and regulation of telomerase at the single cell level.
端粒酶酶通过延长端粒和防止复制性衰老，使大多数人类癌细胞能够无限增殖。尽管端粒酶在癌症生物学中具有至关重要的作用，但在单个细胞中检测端粒酶活性和表达的挑战阻碍了研究端粒酶表达和功能在异质细胞群体中的模式的能力。虽然存在确定端粒酶表达和功能的敏感检测方法，但这些方法已被证明难以在单细胞水平上实施。在这里，我们验证了原位 RNAscope 检测端粒酶 TERT mRNA 的方法，并将该检测方法与我们最近描述的用于原位检测端粒延伸的 TSQ1 方法相结合。这种方法能够在群体中的单个细胞内检测 TERT 表达、端粒长度和端粒延伸。使用该检测方法，我们表明在 HeLa 细胞群体中观察到的异质端粒延伸部分是由 TERT 基因的可变表达驱动的。此外，我们表明，一些 TERT 阳性细胞中未检测到可检测的端粒延伸是端粒庇护复合物抑制的结果。这种组合检测方法为在单细胞水平上理解端粒酶的综合表达、功能和调控提供了一种新方法。
