Acknowledgments
This work was supported by grants from the Spanish Ministry of Economy and Competitiveness, Plan Nacional de I + D + I 2008- 2011, Plan Estatal de I + D + I 2013–2016, ISCIII (Fis: PI12/00137, PI15/00045, RTICC: RD12/0036/0028) co- funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS- 6844 and CTS-1848), and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010, PI-0306-2012 and PI-0096-2014).
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Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_6, © Springer Science+Business Media New York 2017
Chapter 6
Detection of Dysfunctional Telomeres in Oncogene-Induced Senescence
Priyanka L. Patel and Utz Herbig
Abstract
Expressing oncogenes in normal somatic human cells leads to cellular senescence after just a few cell division cycles. In cells that are more resistant to culture stresses, such as human dermal fi broblasts, this oncogene- induced senescence (OIS) is a result of a DNA damage response (DDR) that is activated due to the forma- tion of DNA lesions at both non-telomeric and telomeric DNA sequences. DNA lesions can be visualized as DDR foci by immunofl uorescence microscopy using antibodies against a number of DDR factors, including ϒ-H2AX and 53BP1. Over time and as cells remain arrested in OIS, non-telomeric DDR foci progressively become resolved, while telomeric DDR foci, also called dysfunctional telomeres, persist.
Here we describe a protocol to detect dysfunctional telomeres in cultured human cells, to monitor a tem- poral enrichment of dysfunctional telomeres in cells that had undergone OIS, and to detect dysfunctional telomeres in paraffi n-embedded and formalin-fi xed human tissue.
Key words Telomere dysfunction , TIF , Oncogene , Senescence , FISH , Immunofl uorescence , Cancer , Cell culture , Tissue
1 Introduction
Vertebrate telomeres consist of repetitive hexanucleotide (TTAGGG)n repeats that, together with a protein complex called shelterin, form cap-like structures at the ends of linear chromo- somes [ 1 ]. A primary function of telomeres is to prevent a cell from sensing linear chromosome ends as breaks in the DNA, thereby suppressing illegitimate DNA repair events and end-to- end chromosome fusions. In cells that lack detectable telomerase activity, such as normal somatic human cells, telomeres erode with every cell division cycle and once critically short they become dys- functional and trigger a proliferative arrest termed telomere dysfunction- induced cellular senescence (TDIS) [ 2 ]. Dysfunctional telomeres can be visualized by immunofl uorescence microscopy of cells immunostained using antibodies against DDR factors such as ϒ-H2AX or 53BP1, combined with fl uorescence in situ
hybridization (FISH) using a fl uorophore-labeled peptide nucleic acid (PNA) that is complementary to telomeric repeats [ 3 ].
Co-localizations between DDR foci and telomeric signals are called telomere dysfunction-induced DNA damage foci (TIF) and indi- cate that a telomere had become dysfunctional [ 4 ].
We previously demonstrated that introducing oncogenes such as HRas G12V and BRaf V600E , using retroviruses, causes cells to enter OIS approximately 1 week following transduction. Cells entered OIS with numerous DDR foci, most of which did not co-localize with telomeric repeats. As cells remained senescent, non-telomeric DDR foci were resolved while telomeric DDR foci, or TIF, per- sisted. Due to the persistence of TIF, cells remained stably arrested for a period of up to 30 days. TIF could also be detected in benign human tumors and cancer precursor lesions, suggesting that TDIS stabilizes cellular senescence also in vivo [ 5 ]. Here in this protocol, we describe methods to effi ciently detect dysfunctional telomeres in cultured human cells, monitor their enrichment as cells undergo and remain in OIS, and detect cells displaying TIF in human paraffi n- embedded and formalin-fi xed tissue sections.
2 Materials
Prepare all solutions using deionized ultrapure water and store at indicated storage conditions.