Stable Cellular Senescence Is Associated with Persistent DDR Activation

<div><p>The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged <i>in vitro</i> culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability <i>in vitro,</i> due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.</p></div

Candida spp. in oral cancer and oral precancerous lesions

To assess the presence of Candida spp. in lesions of the oral cavity in a sample of patients with precancer or cancer of the mouth and evaluate the limitations and advantages of microbiological and histological methods, 103 subjects with precancerous or cancerous lesions and not treated were observed between 2007 and 2009. The presence of Candida in the lesions was analyzed by microbiological and histological methods. Cohen's k statistic was used to assess the agreement between culture method and staining techniques. Forty-eight (47%) patients had cancer and 55 (53%) patients had precancerous lesions. Candida spp. were isolated from 31 (30%) patients with cancerous lesions and 33 (32%) with precancerous lesions. C. albicans was the most frequent species isolated in the lesions. The k value showed a fair overall agreement for comparisons between culture method and PAS (0.2825) or GMS (0.3112). This study supports the frequent presence of Candida spp. in cancer and precancerous lesions of the oral cavity. Both microbiological investigations and histological techniques were reliable for detection of Candida spp. It would be desirable for the two techniques to be considered complementary in the detection of yeast infections in these types of lesions

Apparent differential DNA damage response activation during IR-induced cellular senescence correlates with cell survival.

<p><b>a–b.</b> BJ and IMR-90 cells were irradiated with 20 Gy and stained for 53BP1, as a DDR marker, three, ten and thirty days later. Bar graphs show the quantification of 53BP1-positive cells and number of 53BP1 foci per cell ± s.e.m. Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). <b>c.</b> BJ and IMR-90 cells were irradiated or not with 20 Gy. Graphs show the average cell number ± s.d. at different time points, in irradiated cells and non-irradiated controls (* p-value <0.05; ** p-value <0.01).</p

DDR is detectable months after establishment of telomere-initiated cellular senescence.

<p><b>a.</b> In BJ cells, DDR, in the form of γH2AX foci, is detectable long time (up to three months) after establishment of telomere-initiated cellular senescence. Scale bar, 40 µm. <b>b.</b> Bar graph shows the fraction of γH2AX foci-positive cells ± s.e.m. before (young and pre-sen) and at the indicated time points after senescence establishment. Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). <b>c.</b> Bar graph shows the average number of γH2AX foci ± s.e.m. per cell at the indicated time points before and after senescence establishment (*** p-value <0.001; * p-value <0.05).</p

Telomere-initiated senescent cells retain active DDR foci for years after senescence establishment.

<p><b>a.</b> DDR, in the form of ATM pS1981 foci co-localizing with 53BP1 and γH2AX foci, is detectable three years after senescence establishment. Scale bar, 10 µm. Below, bar graphs show the percentage of cells positive ± s.e.m. for the indicated DDR markers, in senescent (sen), early passage proliferating (prol) or telomerized proliferating (tel) skin fibroblasts from two independent centenarian donors (cen2 and cen3). Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). <b>b.</b> SA-β-gal staining of the two batches, cen2 and cen3, is shown together with the percentage of BrdU-positive cells. Scale bar, 100 µm.</p

DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs

The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs

From "cellular" RNA to "smart" RNA: multiple roles of RNA in genome stability and beyond

Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA

Telomere damage promotes vascular smooth muscle cell senescence and immune cell recruitment after vessel injury.

Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of multiple vascular pathologies, including following neointimal formation after injury and atherosclerosis. However, human VSMCs in advanced atherosclerotic lesions show reduced cell proliferation, extensive and persistent DNA damage, and features of premature cell senescence. Here, we report that stress-induced premature senescence (SIPS) and stable expression of a telomeric repeat-binding factor 2 protein mutant (TRF2T188A) induce senescence of human VSMCs, associated with persistent telomeric DNA damage. VSMC senescence is associated with formation of micronuclei, activation of cGAS-STING cytoplasmic sensing, and induction of multiple pro-inflammatory cytokines. VSMC-specific TRF2T188A expression in a multicolor clonal VSMC-tracking mouse model shows no change in VSMC clonal patches after injury, but an increase in neointima formation, outward remodeling, senescence and immune/inflammatory cell infiltration or retention. We suggest that persistent telomere damage in VSMCs inducing cell senescence has a major role in driving persistent inflammation in vascular disease

Inhibition of DNA damage response at telomeres improves the detrimental phenotypes of Hutchinson–Gilford Progeria Syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is a genetic disorder characterized by premature aging features. Cells from HGPS patients express progerin, a truncated form of Lamin A, which perturbs cellular homeostasis leading to nuclear shape alterations, genome instability, heterochromatin loss, telomere dysfunction and premature entry into cellular senescence. Recently, we reported that telomere dysfunction induces the transcription of telomeric non-coding RNAs (tncRNAs) which control the DNA damage response (DDR) at dysfunctional telomeres. Here we show that progerin-induced telomere dysfunction induces the transcription of tncRNAs. Their functional inhibition by sequence-specific telomeric antisense oligonucleotides (tASOs) prevents full DDR activation and premature cellular senescence in various HGPS cell systems, including HGPS patient fibroblasts. We also show in vivo that tASO treatment significantly enhances skin homeostasis and lifespan in a transgenic HGPS mouse model. In summary, our results demonstrate an important role for telomeric DDR activation in HGPS progeroid detrimental phenotypes in vitro and in vivo