Detection of Dysfunctional Telomeres in Oncogene-Induced Senescence

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 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.

Telomeres and replicative cellular aging of the human placenta and chorioamniotic membranes

“…In the final analysis, the buildup of short telomeres is expressed in telomere dysfunction, which is captured by TIF 7 – 10 . We found it essential, therefore, to examine the relation in the placenta and CAMs between TL parameters and TIF.…”

“…We propose that these ‘replicative aging’ hypotheses of the placenta and CAMs require revisions based on the following premise: The shortest telomeres 5 , 6 , expressed in dysfunctional telomeres 7 – 10 and their buildup, rather than mTL, are the indicators of telomere-mediated aging of the placenta and CAMs, as their biological utility draws to an end. We proceeded as follows to test this idea: First, we obtained samples at the end of normal (full term) pregnancies from the placenta, the CM and AM, UCB and maternal blood from mother-newborn pairs.…”

“…In these samples and all other samples collected in this study, we (a) measured the mean length of the terminal restriction fragments (TRFs), generated by Southern blotting (SB) 11 , and (b) measured and tallied the TRFs by the newly-developed Telomere Shortest Length Assay (TeSLA) 12 . Moreover, we quantified telomere dysfunction-induced DNA damage foci (TIF) 10 in all placental and CAM samples. Second, we measured TL parameters by SB and TeSLA, and quantified TIF in placenta and CAMs samples obtained after elective abortion at mean gestational age of 18 weeks.…”

Senescence‐associated β‐galactosidase reveals the abundance of senescent CD8+ T cells in aging humans

“…Telomere‐ImmunoFISH was performed as described in (Patel & Herbig, 2017). Briefly, following the blocking step, cells were washed 3 times with PBS (5 min each) and were dehydrated by subsequent submersion in 70%, 90%, and 100% ethanol (3 min each).…”