We assessed prematuresenescence of endothelia using a normal-risk orthotopic mice corneal transplantation model for three times (totally 60 mice). We collected 8, 11, and 10 corneal grafts for these three times. For each time, four corneal grafts from each of the two groups were used for the detection of staining. That is, one cor- neal graft was separated into three pieces, and one pieces were used for the staining of the corneal endothe- lium with trypan blue and alizarin red S. The others were used for staining of SA-β-Gal and immunohisto- chemical analysis of 8-OHdG.
HCMV-infected HELF accumulate high levels of p16. Cel- lular senescence is accompanied by a series of changes that, together, distinguish senescence from quiescence or differen- tiation (8, 11). These changes include altered expression of cell cycle proteins; upregulation of p53, p16, and p21; and the accumulation of SA-␤-Gal. Earlier reports showed that by 24 h after HCMV infection the steady-state levels of p53 increase dramatically but its p21 and MDM2 targets are not activated (17, 42). This functional inactivation of p53 could be partly the result of its sequestration into viral replication centers in in- fected-cell nuclei. To define the molecular events underlying these effects of HCMV infection on proliferation, we assayed by Western blot analysis the steady-state levels of the tumor suppressor proteins p53 and pRb and the cdki p16 and p21 at different time points after infection or mock infection. In ac- cord with previous reports, we also observed increases in p53 steady-state levels in HCMV-infected cells (42). Its levels were maximal at 48 h p.i. and then declined but were always higher in the infected cells (Fig. 3); highly phosphorylated pRb, ac- companied by a notable increase in Rb steady-state levels, was induced at the same time point. Moreover, as reported by Chen et al. (12), the abundance of p21 declined over the next 48 h p.i. and remained low through 96 h p.i. compared to that in mock-infected cells. Interestingly, significant increases in the amount of p16 protein in HCMV-infected cells at 72 and 96 h p.i. (4.3- and 4.6-fold, respectively) were observed, while its levels in mock-infected cells remained relatively low. This pro- tein may thus be critical for prematuresenescence induced by HCMV infection.
To establish an auditory cell model of prematuresenescence, we used low doses of H 2 O 2 as a stressor to induce prematuresenescence in HEI-OC1 auditory cells. H 2 O 2 has been used to induce prematuresenescence in other cell types as well, such as vascular endothelial cells  and keratinocytes . In our system, brief H 2 O 2 treatment resulted in delayed cell proliferation in the absence of a remarkable decrease in cell viability (Figure 1A and 1B), suggesting that the cells underwent oxidative stress-induced prematuresenescence. As shown in Figure 1C, the cytoplasm of cells displaying morphological characteristics common to cellular senescence stained intensely for SA-β-gal, a distinct marker of cellular senescence. By contrast, the proportion of BrdU incorpo- rated into newly synthesized DNA significantly decreased. These results support the use of our system as a model for studying prematuresenescence in auditory cells. This report is the first to show that SA-β-gal is useful for staining non-dividing, postmitotic cells like auditory cells.
A yeast model has shown that TRIM32 mutations in- volving the NHL domain introduce conformational changes that impair the interaction properties of the protein, and consequently the ubiquitination process . The most relevant mechanistic studies have been performed in the Trim32 knockout (T32KO) and the knock-in mice carrying the Hutterite mutation (T32KI) [25, 26]. TRIM32, as a ubiquitous E3 ubiquitin ligase, has been demonstrated to promote degradation of sev- eral targets [1, 8, 18, 22, 24, 29, 31, 37], so the absence or abnormal function of TRIM32 due to recessive muta- tions would lead to loss of ubiquitination and accumula- tion of the TRIM32 substrates. E3 small ubiquitin-related modifier (SUMO) ligase (PIAS4)  and N-myc down-regulated protein 2 (NDRG2) have been previously identified as important TRIM32 sub- strates. Overexpression of PIAS4 is implicated in regula- tion of cellular senescence  and TRIM32-deficient primary myoblasts from T32KO mice have been demon- strated to undergo prematuresenescence and impaired myogenesis due to accumulation of PIAS4 . On the other hand, NDRG2 overexpression in C2C12 myoblasts reduces cell proliferation and delayed cell cycle with- drawal during differentiation [14, 31]. Altogether, these results coming from cell and animal models, support the hypothesis that TRIM32 is involved in control of myo- genesis and that prematuresenescence underlies myop- athy in LGMD2H.
Introduction Chronic and debilitating low back pain is a common condition and a huge economic burden. Many cases are attributed to age-related degeneration of the intervertebral disc (IVD); however, age-related degeneration appears to occur at an accelerated rate in some individuals. We have previously demonstrated biomarkers of cellular senescence within the human IVD and suggested a role for senescence in IVD degeneration. Senescence occurs with ageing but can also occur prematurely in response to stress. We hypothesised that stress-induced prematuresenescence (SIPS) occurs within the IVD and here we have investigated the expression and production of caveolin-1, a protein that has been shown previously to be upregulated in SIPS.
Transgenics had early flowering at about 50 days which was associated with a shorter vegetative period. Tobacco plants exhibited successive leaf senescence, starting from the basal leaves and advancing toward the apical leaves. At the filling period, which generally occurs in 20 week- old plants, the T3-1, T3-2 and T3-3 plants had at least two dead basal leaves, whereas WT plants only had one dead basal leaf (Fig. 2C). Changes in transgenic plant phenotype were indicative of prematuresenescence. Meanwhile, the transgenic plants developed new stems at the upper part of the plant. This phenomenon might be related to weak sink strength, which causes a slower pro- gression of senescence in tobacco . The new collateral delayed the death of the whole plant. Tobacco senescence is largely independent of floral development making it a model plant species for research . However, leaf senescence in transgenic plants began during vegetative growth.
Autophagy is a cellular catabolic mechanism that is activated in response to stress conditions, including ultraviolet (UV) irradiation, starvation, and misfolded protein accumulation. Abnormalities in autophagy are associated with several pathologies, including aging and cancer. Furthermore, recent studies have demonstrated that microRNAs (miRNAs) are potent modulators of the autophagy pathway. As a result, the current study aims to elucidate the role of the autophagy- related miRNA miR-23ain the process of photoaging. Experiments demonstrated that the antagomir-mediated inactivation of miR-23a resulted in the stimulation of PUVA- and UVB-depressed autophagy flux and protected human fibroblasts from prematuresenescence. Furthermore, AMBRA1 was identified as a miR-23a target. AMBRA1 cellular levels increased following the introduction of miR-23a antagomirs. And a bioinformatics analysis revealed that the AMBRA1 3’ UTR contains functional miR-23a responsive sequences. Finally, it was also demonstrated that both AMBRA1 overexpression and Rapamycin treatment were both able to rescue fibroblasts from PUVA and UVB irradiation-induced autophagy inhibition, but that these effects could also be mitigated by miR-23a overexpression. Therefore, this study concludes that miR-23a-regulated autophagy is a novel and important regulator of ultraviolet-induced prematuresenescence and AMBRA1 is a rate-limiting miRNA target in this pathway.
Although telomere length is the molecular clock that regulates the number of times a normal cell can divide, several studies have shown that replication-associated telomere attrition is not the only process that can induce cellular senescence. Cells also show many senescence- associated phenotypes as a consequence of physiological insults. This form of senescence is called stress-induced prematuresenescence or SIPS, and occurs independently of telomere length. Several intrinsic factors have been identified that cause SIPS in cells grown in culture. These include suboptimal culture media conditions, high oxygen levels, and the absence of cellular microenvironments. SIPS can also be a result of extrinsic factors including oxidative stress (growing cells in oxygen-rich conditions or exposure to hydrogen peroxide and t-butyl- hydroperoxide), DNA damaging agents (ionizing radiation and chemotherapeutic agents), oncogene hyperactivation or over-expression (Ras, BRAF) or abnormal tumor suppressor levels (p16 INK4a , p53) . The number of agents
, Polmu , Vhl , Dicer  and TGFβ  have been observed to limit tissue culture-induced senescence similar to Gadd45b, where their loss resulted in prematuresenescence. Thus, it is of interest to determine whether there is crosstalk between Gadd45b regulated molecular pathways that protect cells from undergoing tissue culture- induced senescence and molecular pathways regulated by these other proteins. Notably, the observation that Gadd45b loss results in prematuresenescence is in contrast to Gadd45a KO MEFs that were observed to escape senescence (Unpublished data). Thus, it will also be of interest to compare and contrast Gadd45b signaling to Gadd45a signaling in MEFs, where loss of Gadd45a results in escape from tissue cultured senescence. Current research is targeted at addressing these interesting issues.
SIRT1 protects against telomere shortening and erosion (a bio- logical maker of replicative senescence) (53). However, the role of SIRT1 in CS-induced replicative senescence is unclear, although telomere length is a determinant of emphysema susceptibility (54). Furthermore, the lung levels of p16, p21, and p27, as well as SA– β-gal activity, were further increased in Foxo3-deficient mice with emphysema, which was supported by a prior study showing the protection of FOXO3 against cellular senescence (14). Strikingly, Foxo3 deficiency diminished the effect of SRT1720 in attenuating the levels of p21 and p16 as well as SA–β-gal activity in emphyse- matous lungs, indicative of the requirement of FOXO3 for SIRT1’s protection against SIPS. Importantly, deletion of p21 significantly ameliorated CS-induced airspace enlargement and lung function decline. Both CS and sirtinol induced an increase in SA–β-gal activity in mouse lung, which was significantly attenuated by p21 deficiency. Hence, SIRT1 activation downregulated SIPS through FOXO3/p21 pathway, thereby protecting against emphysema. In addition to FOXO3, recent studies have demonstrated the involve- ment of other developmental and senescence-related genes, such as Wnt/β-catenin, Notch, Klotho, senescence marker protein-30, and Werner syndrome protein, in the development of emphysema (55–60). However, it remains to be seen whether SIRT1 targets these genes in response to CS exposure.
This study also has some limitations. First, NP cells were scaffold-cultured under normoxic conditions that differ from the physiological conditions in which NP cells are contained in a three-dimensional (3D) environ- ment under hypoxic conditions. Second, two sources of ROS generation exist: endogenic and ectogenic. These two types of ROS may cause different signals in cell biol- ogy. However, the ROS generated by mechanical stress in this study is mainly endogenic. Further studies are needed to verify whether the ectogenic ROS under high- magnitude compression has similar effects on NP cell senescence. Third, although we verified the positive effects of high-magnitude compression on NP cell senes- cence in the rat disc organ culture, the roles of the p38 MAPK pathway and ROS generation in this process were not further investigated using their inhibitors. However, our previous study has demonstrated that inhibition of the p38 MAPK pathway attenuates high- magnitude compression-induced NP cell senescence in the porcine disc organ culture . Additionally, previ- ous studies have demonstrated that inhibition of ROS inhibits disc cell senescence and ageing-related disc degeneration in vitro and in vivo [22, 56, 57]. Therefore, we speculate that either inhibition of the p38 MAPK pathway or suppression of ROS generation can attenuate high-magnitude compression-induced NP cell senescence in the rat disc organ culture.
There have been many studies on the chlorophyll content of cotton leaves assessed with chlorophyll meters  . Chlorophyll content increased in cotton leaves when plants were subjected to drip irrigation below the film . These studies drew inconsistent conclusions about the sensitivity of cotton leaves to N application. Some investigations indicated that the fourth leaf was extremely sensitive to N, while the second leaf was rela- tively insensitive to N, and the first and third leaves varied between the cotton varieties . Qu et al.  found that cauline leaf SPAD values at different positions on two cultivars responded differently to increasing N rate. The first fully-expanded cauline leaf (SL1) was most sensitive, while the fourth fully-expanded leaf (SL4) responded the least. As the N application rate increased, the SL4 and SL1 in the two cultivars converged on a similar SPAD value. The leaves could be well-characterized by the relative positional difference index (PDI), reflecting the greenness difference between SL1 and SL4. PDI can be an ideal indicator of cotton N status. Another study found that leaf SPAD values can also be used to estimate the degree of senescence in cotton leaves . In the study they compared SPAD values on the day of flowering and 35 d after flowering as an in- dicator of chlorophyll content reduction, to measure the rate of reduction, and to track senescence . Wu et al.  obtained the optimum SPAD values at different crop development stages when N was applied in appropri- ate topdressing amount.
Cellular senescence is believed to function as a tumor suppression mechanism, yet the senescence- associated secretory phenotype can promote cancer development and progression by stimulating the proliferation of tumor cells, inducing an epithelial-to- mesenchymal transition or protecting neighboring cells from the effects of chemotherapy . Similarly, CUX1 belongs to this class of cancer genes that can both protect against cancer and promote cancer development and progression depending on physiological context . It remains to be demonstrated whether the DNA repair function of CUX1 contribute to its role as a tumor suppressor, however, it is clear that RAS-driven cancer cells exploit this function of CUX1 to avoid senescence and continue to proliferate in spite of elevated levels of reactive oxygen species .
results showed that expression of both p53 and p16 did not changed at each time points observed (Figure 2A). CoCl 2 -induced senes- cence appears to be independent of these pathways and engage others. Bcl-2 was report- ed to modulate cell cycle progression, favoring a quiescent state over a proliferative state . Therefore, Bcl-2 expression was tested and data showed that its expression increased gradually from the time CoCl 2 added and reached a high level at 24 h and then main- tained this state with the occurrence of cellular senescence occurred up to 72 h (determined by detection of SA-β-gal activity synchronously) (Figure 2A). Similarly, statistical results of co- location of SA-β-gal and Bcl-2 expression re- vealed that 19.2% senescent cells were posi- tive for Bcl-2 at 36 h after CoCl 2 treatment, com- pared with 1.1% at normoxia (Figure 2B). These results reveal that Bcl-2 may be involved in the regulation of hypoxia-induced senescence. Effect of Bcl-2 expression on cellular senes- cence development
SA-β-gal assay measures the activity of a lysosomal β-galactosidase. Therefore, an increase in SA-β-gal could be due to increased enzyme activity or expression. Research suggests that the latter is the case. SA-β-gal activity in senescent dividing cells is owed at least in part to increased levels of lysosomal β-galactosidase mRNA and protein and has been linked to increased lysosome number or activity . Moreover, it is likely that SA-β-gal is not directly linked to senescence because silencing of the Glb1 gene, which encodes lysosomal β-galactosidase, does not alleviate the symptoms of senescence . Thus, we think that rather than senescence we witnessed a continued growth of neurons, in which, as our experiments showed, lysosomes grew in number giving rise to increased SA-β- gal activity. However, it cannot be excluded that during development in vitro neurons can undergo a senescence- like process, which is characterized by similar markers as senescence of dividing cells. Accordingly, we analysed DNA damage in neurons. It is believed that DDR signalling has a causative role in the establishment of cellular senescence . DNA damage is known to be involved in the induction of replicative senescence and prematuresenescence can be caused by such agents as ionizing radiation or DNA damage-inducing drugs [20, 21]. For example, in our previous studies we showed that low doses of doxorubicin added to cancer or normal cells induced SIPS, one of the features of which was increased SA-β-gal [22, 23]. In cortical neurons, however, we demonstrated minimal DNA double-strand damage during a long-term culture in both SA-β-gal positive and negative cells, which implies that neurons acquired SA-β-gal activity without DDR activation. Further, triggering DDR by low doses of doxorubicin did not increase the number of SA-β-gal-positive neurons. Altogether, we can conclude
The accelerated cellular senescence characteristic of G6PD-deficient fibroblasts would seem to represent a clear case of a senescence mechanism based on accumu- lated oxidative DNA damage rather than one involving accelerated telomere attrition . This mechanistic transparency makes these cells an ideal system for addressing the relative role of oxidative stress and tel- omere attrition in cellular senescence. Somewhat surpris- ingly, we found that ectopic expression of hTERT prevented the accelerated senescence of G6PD-deficient cells and led to their immortalization (Fig. 2). The growth rate of hTERT-expressing G6PD-deficient fibroblasts, however, remained slower than that of hTERT-expressing normal fibroblasts (Fig. 2A and 2B), as noted previously (25). This finding suggests that hTERT overexpression may attenuate senescence induction by oxidative stress, but does not suppress the growth defect caused by G6PD- deficiency. To test this hypothesis, we measured the abil- ity of hTERT-expressing G6PD-deficient cells to cope with H 2 O 2 -induced oxidative stress. Similar to results reported by others , we found no difference in stress-induced prematuresenescence (SIPS) between normal and hTERT- expressing normal fibroblasts (Fig. 5). However, hTERT- expressing G6PD-deficient cells became more resistant to H 2 O 2 -induced prematuresenescence (Fig. 5), indicating that the increased sensitivity to oxidative stress in G6PD- deficient cells is prevented by the expression of hTERT. Ectopic expression of hTERT has also been shown previ- ously to immortalize fibroblasts derived from individuals with Ataxia telangiectasia (A-T), Nijimegen breakage syn- drome (NBS), Hutchinson-Gilford progeria syndrome (HGPS), and Werner Syndrome (WS) [32-36]. Although the genetic defects in A-T, NBS, HGPS, WS, and G6PD- deficiency patients are very different, fibroblasts derived from these individuals have one common phenotype: they all undergo accelerated senescence in vitro [25,34,36,37]. The prematuresenescence of mitotic cells derived from A-T, HGPS, and NBS patients has been cor- related with an increased rate of telomere loss [31,34,38], whereas the mechanism responsible for the prematuresenescence of WS and G6PD-deficient fibroblasts appears to be different and has been postulated to reflect the accu-
Cellular senescence is a programmed response triggered in normal cells experiencing various types of stimuli, including telomere erosion [1-3], DNA damage , induction of oncogenes  and oxidative stress . Replicative senescence is a specialized cellular mechanism which occurs following an extended period of proliferation of normal cells driven by excessive telomere erosion and dysfunction [1,7]. Senescent cells remain metabolically active, yet are irreversibly arrested in the cell cycle . They become enlarged and flattened , undergo drastic changes in chromatin structure and gene expression , and as a result, express senescence-associated β- galactosidase activity . Replicative senescence is considered to be protective against malignant transformation since it ceases the extended propagation of cells. Cellular senescence, as an intrinsic mechanism, also acts to prevent proliferation in response to acute stresses, such as DNA damage, this is collectively referred to as stress-induced prematuresenescence (SIPS) . Mechanistically, inducers of replicative senescence and SIPS elicit the damage signal and trigger two major tumor suppressive pathways, p53 and p16. Activation of p53, and subsequently p21 (CIP1, WAF1), is associated with the DNA damage response (DDR) pathway, mediated by ATM/ATR and Chk1/Chk2 kinases, which can post-translationally stabilize p53, leading to its activation . In turn, transcriptional activation of the p53 target protein, p21, reduces Cdk2 kinase activity .
Accordingly, our studies revealed that stress-induced prematuresenescence (SIPS) of vascular smooth muscle cells (VSMCs) induced by doxorubicin or H 2 O 2 , correlates with increased level of DSB and ROS. On the other hand, both SIPS and replicative senescence were accompanied by diminished expression of NOX4. Moreover, inhibition of NOX activity or decrease of NOX4 expression led to permanent growth arrest of VSMCs and secretion of interleukins and VEGF. Interestingly, cells undergoing senescence due to NOX4 depletion neither acquired DSB nor activated DNA damage response. Instead, transient induction of the p27, upregulation of HIF-1alpha, decreased expression of cyclin D1 and hypophosphorylated Rb was observed. Our results showed that lowering the level of ROS-producing enzyme - NOX4 oxidase below physiological level leads to cellular senescence of VSMCs which is correlated with secretion of pro-inflammatory cytokines. Thus the use of specific NOX4 inhibitors for pharmacotherapy of vascular diseases should be carefully considered.
Summarizing, we have shown that curcumin did not protect cells building the vasculature from senescence. However, it was able to upregulate the level of sirtuins 1, 3, 5, 6 and 7. Curcumin might activate/upregulate sirtuins by AMPK activation elicited by ROS elevation and ATP reduction. Our results suggest that the beneficial anti-aging effect attributed to curcumin is not caused by postponing cellular senescence and we propose that it could be due to the activation of sirtuins. However, even though curcumin did not postpone cellular senescence in vitro, it cannot be excluded that it may act differently in vivo. One of the activities of sirtuins is the epigenetic regulation of the activity of NFκB. This transcription factor, responsible for inflammation and associated with both cellular senescence and aging [39, 40], is the most recognized anti- inflammatory molecular target of curcumin. Curcumin inhibits NFκB via its effect on the kinase necessary to dissociate IκB (inhibitor of kappa B). Sirtuin 6 is able to deacetylate histone H3 at lysine 9 at the promoter of RELA (component of NFκB), causing inhibition of transcription and loss of NFκB activity . Taking into account that curcumin increased the level of sirtuin 6, it can be assumed that there is another way of NFκB inhibition by curcumin. Moreover, it has been shown that other factors, including resveratrol, cilostazol, paeonol, statins and hydrogen sulfide, could protect cells from senescence by regulating sirtuin 1 [42 - 46]. Activators of sirtuins are considered as potential anti-aging and CVD protecting factors . Sirtuin 1 is responsible for the beneficial effect of mild physical activity [48, 49] and supplementation of the diet with curcumin improved the effects of physical activity . On the other hand, downregulation of sirtuins caused prematuresenescence . Furthermore, sirtuins are considered as markers of frailty, which is one of the most important features of aging . As it has been proposed by Rattan and Ali , repeated mild heat stress (RMHS) could act as an anti-aging factor. The authors suggested that curcumin, as a hormetin, could support the effect of
of GQP. In the present study, we investigated the anti-aging activities of GQP in WI-38 cells treated by H 2 O 2 and the related mechanisms. We found the optimal concentration of H 2 O 2 treatment in inducing prematuresenescence in WI-38 cells, and established H 2 O 2 -induced pre- mature senescent WI-38 cell model. H 2 O 2 treat- ment at 100 μmol/L could cause significant changes in cellular morphology and cytoplas- mic inclusions, which were typical senescent features. Our results showed that GQP could not only dramatically restore the altered mor- phology, but also significantly increase the cell viability and proliferation. The protective effects of GQP against H 2 O 2 -induced cellular senes- cence were further confirmed by the detection of senescence markers. SA-β-gal activity is the most widely used indicator for cellular senes- cence [34, 35]. Our results showed that H 2 O 2 led to an increase in SA-β-gal activity in WI-38 cells, which could be inhibited by GQP treatment.