Priority Research Paper Volume 1, Issue 7 pp 608—621

Figure 2. Replicative senescence and DNA damage signaling independent up-regulation of ISG15 expression in cells with short telomeres. (A) Short and long-term expression of hTERT rescued cells from replicative senescence. BJ cells with short telomeres (BJ-80 and BJ13-141) exhibited significant increases in the number of SA-β-Gal positive cells; whereas, the cells with long telomeres (BJ-18) did not show SA-β-Gal staining. Exogenous telomerase rapidly eliminated senescent cells (BJ13-141+6H, when only the shortest telomeres had been lengthened) as well as after bulk telomere elongation had occurred (BJ13-141+53H). Rare fields with an SA-β-Gal staining positive cell were selected for the last two images to validate the staining procedure. The number in each image is a key to the cell lines used in B-D. (B) γ-H2AX staining shows that exogenous hTERT rapidly eliminates DNA damage signalling due to short telomeres. Approximately 500 nuclei of each cell line were analyzed using Metasystems software (Metasystems, Germany). (C) Western blot shows that p21, a transcriptional target of DNA damage-induced p53 signaling, rapidly disappeared following the introduction of telomerase to elongate the shortest telomeres. (D) Q-PCR showing that ISG15 expression remained high in BJ cells rescued from replicative senescence/DNA damage signaling after only a few doublings in the presence of exogenous telomerase when telomeres were still short (ISG15, column 4), while elongation of the telomeres after 53 doublings led to decreased expression (ISG15, column 5). In contrast, elimi-nating replicative senescence/DNA damage following a short exposure to telomerase caused a decrease in the expression of agrin (agrin, column 4). Agrin thus did not meet our criteria for telomere length regulation, since its increase in old cells (agrin, columns 2&3) is secondary to senescence and/or DNA damage (column 4). (E) BJ cells overexpressing hTERT and having long telomeres express low levels of ISG15.