Detection of Dysfunctional Telomeres in Oncogene-Induced Senescence.
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 fibroblasts, this oncogene-induced senescence (OIS) is a result of a DNA damage response (DDR) that is activated due to the formation of DNA lesions at both non-telomeric and telomeric DNA sequences. DNA lesions can be visualized as DDR foci by immunofluorescence 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 temporal enrichment of dysfunctional telomeres in cells that had undergone OIS, and to detect dysfunctional telomeres in paraffin-embedded and formalin-fixed human tissue.
Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School-Cancer Center, Rutgers Biomedical and Health Sciences, 205 South Orange Avenue, Newark, NJ, 07103, USA.
Priyanka L
PL
Patel
Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School-Cancer Center, Rutgers Biomedical and Health Sciences, 205 South Orange Avenue, Newark, NJ, 07103, USA. herbigut@njms.rutgers.edu.
Utz
U
Herbig
D015415:Biomarkers; D045744:Cell Line, Tumor; D016922:Cellular Senescence / Q000235:genetics*; D004249:DNA Damage; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009857:Oncogenes / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D016615:Telomere / Q000235:genetics*; D014161:Transduction, Genetic
D015415:Biomarkers
Cancer; Cell culture; FISH; Immunofluorescence; Oncogene; Senescence; TIF; Telomere dysfunction; Tissue