Senescence-associated Heterochromatin Foci Research Articles

Downregulation of Histone H3.3 Induces p53-Dependent Cellular Senescence in Human Diploid Fibroblasts

Cellular senescence is an irreversible growth arrest that acts as a barrier to cancer initiation and progression. Histone alteration is one of the major events during replicative senescence. However, little is known about the function of H3.3 in cellular senescence. Here we found that the downregulation of H3.3 induced growth suppression with senescence-like phenotypes such as senescence-associated heterochromatin foci (SAHF) and β-galactosidase (SA-β-gal) activity. Furthermore, H3.3 depletion induced senescence-like phenotypes with the p53/p21-depedent pathway. In addition, we identified miR-22-3p, tumor suppressive miRNA, as an upstream regulator of the H3F3B (H3 histone, family 3B) gene which is the histone variant H3.3 and replaces conventional H3 in active genes. Therefore, our results reveal for the first time the molecular mechanisms for cellular senescence which are regulated by H3.3 abundance. Taken together, our studies suggest that H3.3 exerts functional roles in regulating cellular senescence and is a promising target for cancer therapy.

Unraveling the association between major depressive disorder and senescent biomarkers in immune cells of older adults: a single-cell phenotypic analysis.

Background: Little is known about the prevalence of cellular senescence among immune cells (i.e., immune cells expressing senescence markers, iSCs) nor is there a gold-standard to efficiently measure iSCs. Major depressive disorder (MDD) in older adults has been associated with many hallmarks of senescence in whole blood, leukocytes, and plasma, supporting a strong connection between iSCs and MDD. Here, we investigated the prevalence and phenotype of iSCs in older adults with MDD. Using a single-cell phenotypic approach, circulating immune cells were examined for iSC biomarkers and their relationship to depression and inflammation. Results: PBMCs from older adults with MDD (aged 69.75 ± 5.23years) and healthy controls (aged 71.25 ± 8.8years) were examined for immune subset distribution and senescence biomarkers (i.e., lack of proliferation, senescence-associated heterochromatin foci (SAHF), and DNA damage). Dual-expression of SAHF and DNA damage was categorized by low, intermediate, and high expression. A significant increase in the number of high expressing total PBMCs (p = 0.01), monocytes (p = 0.008), a trending increase in the number of high expressing CD4 T cells (p = 0.06) was observed overall in those with MDD. There was also a significantly lower proportion of intermediate expressing cells in monocytes and CD4 T cells in MDD (p = 0.01 and p = 0.05, respectively). Correlation analysis revealed associations between iSCs and mRNA expression of factors related to SASP and immune cell function. Conclusion: MDD is associated with increased senescent cell biomarkers in immune cell populations delineated by distinct levels of SAHF and DNA damage. Inflammatory markers might serve as potent indicators of iSC burden in MDD.

Indole-3-Carboxylic Acid Enhanced Anti-cancer Potency of Doxorubicin via Induction of Cellular Senescence in Colorectal Cells

Background: Colorectal cancer (CRC) is the most common gastrointestinal malignancy. Doxorubicin (DOX) is a widely utilized chemotherapy drug, but its efficacy is limited due to dose-dependent toxicity. Here, we aim to explore the effect of indole-3-carboxylic acid on DOX-induced senescence of CRC. Methods: Healthy adult rats and aged rats were compared in terms of their metabolites and functions through non-targeted metabolomics. LS180 cells were treated with DOX to induce senescence, followed by indole-3-carboxylic acid. The effects of this combination were evaluated in xenograft tumor mice. Cell viability, proliferation, and cell cycle were assessed with the Cell Counting Kit-8, colony formation assays, and flow cytometry. The levels of senescence-associated heterochromatin foci (SAHF) were detected by immunofluorescence. Senescence-associated-beta-galactosidase (SA-β-gal) expression was assessed by SA-β-gal staining and immunohistochemistry. Western blot was used to detect the expression of p21 and p53. Results: Compared to healthy adult rats, the serum metabolome in aging rats was altered, and the abundance of indole metabolites, including indoxyl sulfate, indole-3-carboxylic acid, and indole-5-carboxylic acid, was decreased significantly. In LS180 cells, indole-3-carboxylic acid amplified DOX-induced cell senescence, inhibiting cell proliferation and promoting cell cycle arrest. It also boosted DOX-triggered upregulation of SA-β-gal, SAHF, and p21. In nude mice, indole-3-carboxylic acid increased the inhibitory effect of DOX on xenograft tumors. Conclusion: Indole-3-carboxylic acid enhances the cellular senescence and growth arrest induced by DOX, suppressing mouse tumor growth. These findings suggest that a combined treatment of indole-3-carboxylic acid and DOX could be an effective strategy for CRC treatment.

Senescence-Associated Heterochromatin Foci Suppress γ-H2AX Focus Formation Induced by Radiation Exposure.

DNA damage is induced by both endogenous and exogenous factors. Repair of DNA double-strand break (DSB), a serious damage that threatens genome stability, decreases with senescence. However, the molecular mechanisms underlying the decline in DNA repair capacity during senescence remain unclear. We performed immunofluorescence staining for phosphorylated histone H2AX (γ-H2AX) in normal human fetal lung fibroblasts and human skin fibroblasts of different ages after chronic irradiation (total dose, 1 Gy; dose rate, 1 Gy/day) to investigate the effect of cellular senescence and organismal aging on DSB repair. Accumulation of DSBs was observed with cellular senescence and organismal aging, probably caused by delayed DSB repair. Importantly, the formation of γ-H2AX foci, an early event in DSB repair, is delayed with cellular senescence and organismal aging. These results suggest that the delay in γ-H2AX focus formation might delay the overall DSB repair. Interestingly, immediate γ-H2AX foci formation was suppressed in cells with senescence-associated heterochromatin foci (SAHF). To investigate the relationship between the γ-H2AX focus formation and SAHF, we used LiCl to relax the SAHFs, followed by irradiation. We demonstrated that LiCl rescued the delayed γ-H2AX foci formation associated with cellular senescence. This indicates that SAHF interferes with γ-H2AX focus formation and inhibits DSB repair in radiation-induced DSB. Our results suggest that therapeutic targeting of SAHFs have potential to resolve DSB repair dysfunction associated with cellular senescence.

Mechanisms of Aging Vary with Developmental Stages in the Human Hematopoietic Trajectory

Background : Hematopoietic stem and progenitor cells (HSPCs) undergo functional changes with age. An unresolved issue is whether aging is induced by a shift in intrinsic state in which all HSPCs undergo coordinated changes in functional potential, or by clonal evolution of a senescent population.Identification of senescent cells in human hematopoiesis poses another challenge. In this study, we have identified lineage-specific, senescence-associated pathways at different developmental stages of HSPCs and have developed an “aging signature HSC” for non-primed human HSCs. Methods: HSPCs (CD34+ cells) were isolated from the bone marrow of healthy human subjects (old = >60 years; young = 21 to <35 years). Single-cell RNA-sequencing (scRNAseq) studies and downstream analysis were performed using different R packages for normalization, dimension reduction, clustering, classification, pseudotime ordering, and developmental trajectory analyses. Gene Set Enrichment Analysis, GSEA (version 4.3.2., Broad Institute, Inc) was applied to identify pathways and processes that were coordinately up- or down-regulated with aging. Results: HSPCs (CD34+ cells) were derived from 13 healthy human subjects (old subjects n = 6; young subjects n = 7). There were 26,076 genes identified. The HSPCs were ordered and classified along their developmental trajectory during differentiation processes from (1) hematopoietic stem cells (HSC) to (2) myeloid-lymphoid progenitors (MLP), (3) megakaryocyte-erythrocyte progenitors (MEP), (4) granulocytic-monocytic progenitors (GMP), (5) granulocyte- (G), and (6) monocyte- (Mono) progenitors, (7) dendritic cell (DC), (8) erythroid progenitors (E), as well as various subsets of lymphocytes (Ly1 to Ly4). The results are depicted in Figure 1A. Remarkable are the increases in HSCs (p < 0.05) and in Ly4 (p < 0.001) in old subjects. The Kolmogorov-Smirnov test showed a significant delay in the developmental “pseudotimes” of HSCs (p < 0.001) and of Ly4 (p < 0.05). We then compared the differences in GSEA between old and young subjects in each of the following clusters: (A) whole HSPCs; (B) HSPCs along myeloid pathway (mye-HSPC); (C) HSC-MLP-MEP; (D) HSC. The major findings relevant to aging are summarized in Fig. 1B. Pathways that are significantly increased in the old subjects and throughout all comparisons include: “Formation of senescence associated heterochromatin foci”, “Aging signature HSC”, “DNA damage telomere stress induced senescence”, and “TNFR1 induced NFKB signaling pathway”. “Hypoxia”, “P53 pathway”, “IL2 signaling”, “TNFA signaling via NFKB” were elevated upon enrichment of the primitive HSC population, while other pathways decreased: “Senescence associated secretory phenotype (SASP)”, “DNA methylation”. “Cell cycle” and “DNA repair” are consistently more abundant in young subjects. We analyzed the 512 cells in the HSC cluster with higher resolution and were able to identify a sub-cluster “MPP2A” that is significantly increased in old subjects (4.5% of all HSPCs and 28.5% of HSCs, versus 1.9% and 12.9% respectively in young subjects; p < 0.005). The results are depicted in the highlighted circles in Fig. 1A. This population is quiescent and is highly enriched in “aging signature HSC” in old subjects (p <0.0001). Prominent examples of these signature genes within this list are shown in Fig. 1C). Further along the trajectory, a unique population of B cells “Ly4” was found predominantly in old subjects. The transcriptome profile Ly4 is similar to immature and age-associated B cells (ABCs). This cluster of lymphoid cells may represent the precursors of ABCs described thus far only in peripheral blood, providing another evidence that the aging mechanisms at various differentiation stages are different. Conclusions: In this study, we have defined specific pathways that distinctly separate aging HSCPs from young ones, and shown that the definition of “aging signature” varies with developmental stages. The accumulation a small cluster in the non-primed HSC compartment, with “aging signature HSC” and cell cycle arrest in old subjects indicates that aging of human hematopoiesis is induced by clonal evolution. The abundance of a unique population among lymphoid precursors with expression profiles similar to ABCs demonstrates that lymphoid and myeloid progenitors age differently.

Doxorubicin-induced senescence promotes resistance to cell death by modulating genes associated with apoptotic and necrotic pathways in prostate cancer DU145 CD133+/CD44+ cells

Cancer stem cells (CSCs) are the most important cause of cancer treatment failure. Traditional cancer treatments, such as chemotherapy and radiotherapy, damage healthy cells alongside malignant cells, leading to severe adverse effects. Therefore, inducing cellular senescence without triggering apoptosis, which further damages healthy cells, may be an alternative strategy. However, there is insufficient knowledge regarding senescence induction in CSCs that show resistance to treatment and stemness properties. The present study aims to elucidate the effects of senescence induction on proliferation, cell cycle, and apoptosis in prostate CSCs and non-CSCs. Prostate CSCs were isolated from DU145 cancer cells using the FACS method. Subsequently, senescence induction was performed in RWPE-1, DU145, prostate CSCs, and non-CSCs by using different concentrations of Doxorubicin (DOX). Cellular senescence was detected using the senescence markers SA-β-gal, Ki67, and senescence-associated heterochromatin foci (SAHF). The effects of senescence on cell cycle and apoptosis were evaluated using the Muse Cell Analyzer, and genes in signaling pathways associated with the apoptotic/necrotic pathway were analyzed by real-time PCR. Prostate CSCs were isolated with 95.6 ± 1.4% purity according to CD133+/CD44+ characteristics, and spheroid formation belonging to stem cells was observed. After DOX-induced senescence, we observed morphological changes, SA-β-gal positivity, SAHF, and the lack of Ki67 in senescent cells. Furthermore; we detected G2/M cell cycle arrest and downregulation of various apoptosis-related genes in senescent prostate CSCs. Our results showed that DOX is a potent inducer of senescence for prostate CSCs, inhibits proliferation by arresting the cell cycle, and senescent prostate CSCs develop resistance to apoptosis.

Contribution of viral and bacterial infections to senescence and immunosenescence.

Cellular senescence is a key biological process characterized by irreversible cell cycle arrest. The accumulation of senescent cells creates a pro-inflammatory environment that can negatively affect tissue functions and may promote the development of aging-related diseases. Typical biomarkers related to senescence include senescence-associated β-galactosidase activity, histone H2A.X phosphorylation at serine139 (γH2A.X), and senescence-associated heterochromatin foci (SAHF) with heterochromatin protein 1γ (HP-1γ protein) Moreover, immune cells undergoing senescence, which is known as immunosenescence, can affect innate and adaptative immune functions and may elicit detrimental effects over the host's susceptibility to infectious diseases. Although associations between senescence and pathogens have been reported, clear links between both, and the related molecular mechanisms involved remain to be determined. Furthermore, it remains to be determined whether infections effectively induce senescence, the impact of senescence and immunosenescence over infections, or if both events coincidently share common molecular markers, such as γH2A.X and p53. Here, we review and discuss the most recent reports that describe cellular hallmarks and biomarkers related to senescence in immune and non-immune cells in the context of infections, seeking to better understand their relationships. Related literature was searched in Pubmed and Google Scholar databases with search terms related to the sections and subsections of this review.

Dynamic 3D genome reorganization during senescence: defining cell states through chromatin.

Cellular senescence, a cell state characterized by growth arrest and insensitivity to growth stimulatory hormones, is accompanied by a massive change in chromatin organization. Senescence can be induced by a range of physiological signals and pathological stresses and was originally thought to be an irreversible state, implicated in normal development, wound healing, tumor suppression and aging. Recently cellular senescence was shown to be reversible in some cases, with exit being triggered by the modulation of the cell's transcriptional program by the four Yamanaka factors, the suppression of p53 or H3K9me3, PDK1, and/or depletion of AP-1. Coincident with senescence reversal are changes in chromatin organization, most notably the loss of senescence-associated heterochromatin foci (SAHF) found in oncogene-induced senescence. In addition to fixed-cell imaging, chromatin conformation capture and multi-omics have been used to examine chromatin reorganization at different spatial resolutions during senescence. They identify determinants of SAHF formation and other key features that differentiate distinct types of senescence. Not surprisingly, multiple factors, including the time of induction, the type of stress experienced, and the type of cell involved, influence the global reorganization of chromatin in senescence. Here we discuss how changes in the three-dimensional organization of the genome contribute to the regulation of transcription at different stages of senescence. In particular, the distinct contributions of heterochromatin- and lamina-mediated interactions, changes in gene expression, and other cellular control mechanisms are discussed. We propose that high-resolution temporal and spatial analyses of the chromatin landscape during senescence will identify early markers of the different senescence states to help guide clinical diagnosis.

Fisetin Attenuates Cellular Senescence Accumulation During Culture Expansion of Human Adipose-Derived Stem Cells.

Mesenchymal stem cells (MSCs) have long been viewed as a promising therapeutic for musculoskeletal repair. However, regulatory concerns including tumorgenicity, inconsistencies in preparation techniques, donor-to-donor variability, and the accumulation of senescence during culture expansion have hindered the clinical application of MSCs. Senescence is a driving mechanism for MSC dysfunction with advancing age. Often characterized by increased reactive oxygen species, senescence-associated heterochromatin foci, inflammatory cytokine secretion, and reduced proliferative capacity, senescence directly inhibits MSCs efficacy as a therapeutic for musculoskeletal regeneration. Furthermore, autologous delivery of senescent MSCs can further induce disease and aging progression through the secretion of the senescence-associated secretory phenotype (SASP) and mitigate the regenerative potential of MSCs. To alleviate these issues, the use of senolytic agents to selectively clear senescent cell populations has become popular. However, their benefits to attenuating senescence accumulation in human MSCs during the culture expansion process have not yet been elucidated. To address this, we analyzed markers of senescence during the expansion of human primary adipose-derived stem cells (ADSCs), a population of fat-resident MSCs commonly used in regenerative medicine applications. Next, we used the senolytic agent fisetin to determine if we can reduce these markers of senescence within our culture-expanded ADSC populations. Our results indicate that ADSCs acquire common markers of cellular senescence including increased reactive oxygen species, senescence-associated β-galactosidase, and senescence-associated heterochromatin foci. Furthermore, we found that the senolytic agent fisetin works in a dose-dependent manner and selectively attenuates these markers of senescence while maintaining the differentiation potential of the expanded ADSCs.

Protection against Microglia Senescence by the Dietary Supplement Dekosilhue® in BV2 Cells: A New Perspective for Obesity and Related Complications

Growing evidence indicates chronic low-grade systemic inflammation as a major pathophysiological mechanism of obesity. Systemic inflammation provokes an immune response in the brain through the activation of microglia that results in the development of neuroinflammation, cellular senescence, and occurrence of neurological dysfunction. In the efforts to identify an innovative intervention with potential efficacy on obesity and associated complications, our aim was to study the capability of the dietary supplement Dekosilhue® (DKS), successfully used for improving the control of body weight, to attenuate microglia senescent phenotype. Microglia senescence was induced by intermittent stimulation of BV2 cells with LPS 500 ng/mL every 72 h for 4 h/day, over a period of 10 days. DKS (100 µg/mL) treatment reduced ß-galactosidase activity and expression, the formation of senescence-associated heterochromatin foci to control levels, and increased cell viability of senescent BV2 (2 folds of control). DSK reduced the expression of Nuclear Factor-kB (NF-kB) (20% lower than control), a key molecule involved in the acquisition of the senescence-associated secretory phenotype (SASP). DKS promoted a neuroprotective effect by increasing the cell viability of SH-SY5Y neuronal cells exposed to the senescent BV2 conditioned medium to values of non-senescent cells. In conclusion, DKS attenuated the senescent microglia phenotype, showing senotherapeutic activity that might be further investigated as adjunctive intervention for obesity and obesity-related neurological disturbances.

Abstract 425: Characterization of TKI-induced drug-tolerant persister cells from a patient-derived cell line

Abstract Introduction: Targeted therapies provide clinical benefits in patients harboring oncogene-driven cancers, but relapses invariably occur. Studies on resistance mechanisms are essential to guide the development of next-generation inhibitors. An increasing number of studies focus on an earlier stage of disease evolution. Resistance may develop from the selection of preexisting resistant cells or by adaptation of drug-tolerant persister (DTP) cells. In this context, our study depicts DTP cells able to survive the initial tyrosine kinase inhibitor (TKI) exposure without harboring genetic changes, in order to identify the vulnerabilities existing in this cell state. Materials and Methods: This project focuses on a patient-derived cell line from the prospective MATCH-R clinical trial (NCT0251782). The model is a non-small cell lung cancer harboring EML4-ALK fusion and ALK C1156Y/G1269A mutation, sensitive to the 3rd generation ALK TKI lorlatinib. In vitro, fully resistant cells were generated by long-term lorlatinib exposure and the acquired resistance mechanism resulted from an epithelial-mesenchymal transition (EMT). DTP cells were generated under shorter drug exposures that eradicate sensitive carcinoma cells. Results and Discussion: Carcinoma cell plasticity drives cell transformation towards a phenotypic state, rendering them more tolerant to drugs. To better characterize this phenotype, we first focused on EMT and observed that DTP cells co-harbored epithelial and mesenchymal markers. We noticed that DTP cells exhibited an intracellular reactive oxygen species (ROS) rate making them dependent on protective oxidation-reduction mechanisms against oxidative stress-related damage. We revealed the overexpression of p21 and p27, known to bind to cyclin-CDK complexes and induce cell-cycle arrest. This dormant phenotype of DTP cells was underlined by a global repressive chromatin state evidenced by enrichment in H3K27me3, H3K36me3 and H3K9me3 marks; the latter mark also being a component of senescence-associated heterochromatin foci. In agreement, a high level of SA-β-gal and senescence-associated secretory phenotype components (CXCL10, PAI-1), evidenced that our DTP model displayed a senescence-like phenotype. We quantified an increase in γH2AX DNA damage foci in DTP cells compared to sensitive cells, in line with the concept that TKI treatment may have a role in DNA repair modulation that ultimately promotes new mutations. Together, our data depicted DTP features that might be relevant to identify drug candidates overcoming resistance (HDAC inhibitors, senolytic drugs and DNA repair inhibitors). Conclusion: We demonstrated potential targetable features of DTP cells. A combination of targeted therapies with DTP drug candidates could represent a therapeutic opportunity to improve the depth of response and delay the emergence of resistance in patients. Citation Format: Floriane Brayé, Francesco Facchinetti, Helena Gerber, Juliette Juigné, Giorgia Guaitoli, Jean-Paul Thiery, Santiago Ponce, Benjamin Besse, Ken A. Olaussen, Luc Friboulet. Characterization of TKI-induced drug-tolerant persister cells from a patient-derived cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 425.

Role of Histone Variant H2A.J in Fine-Tuning Chromatin Organization for the Establishment of Ionizing Radiation-Induced Senescence.

Radiation-induced senescence is characterized by profound changes in chromatin organization with the formation of Senescence-Associated-Heterochromatin-Foci (SAHF) and DNA-Segments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-Associated-Secretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in establishing radiation-induced senescence. Primary and genetically-modified lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy. Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry. Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory regions decisively modulates transcription factor recruitment and inflammatory gene expression, resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent H2A.J expression in response to radiation-induced DNA damage, therefore contributing to pro-inflammatory tissue reactions. The fine-tuned incorporation of H2A.J defines the epigenetic landscape for driving the senescence programme in response to radiation-induced DNA damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into DNA-damage triggered epigenetic mechanisms governing the biological processes of radiation-induced injury.

Dexamethasone Induces Senescence-Associated Changes in Trabecular Meshwork Cells by Increasing ROS Levels Via the TGFβ/Smad3-NOX4 Axis.

Glaucoma is a serious complication of glucocorticoid (GC) therapy arising through elevations in intraocular pressure (IOP). Dexamethasone (DEX) is reported to contribute to elevated IOP through different effects on the trabecular meshwork but whether DEX contributes to glaucoma development through the induction of cellular senescence is still unclear. We explored the actions of DEX on transformed human trabecular meshwork cells (HTMCs) using RNA-seq and conducted bioinformatic analyses to determine the affected pathways. Among the 4,103 differentially expressed genes identified in transformed HTMCs treated with 400 nM DEX (2,036 upregulated and 2,067 downregulated genes, respectively), bioinformatic analyses revealed significant enrichment and potential interplay between the transforming growth factor beta (TGFβ)41; signaling and cellular senescence pathways. DEX treatment induced senescence changes in primary and transformed HTMCs as indicated by increases in SA-β-gal positivity, interleukin (IL)-6 secretion, and senescence-associated heterochromatin foci (SAHF) along with selective accumulation of senescence marker p15 and elevations in reactive oxygen species (ROS) levels. Notably, the DEX-induced senescence changes were rescued by treatment with the TGFβ/Smad3 pathway inhibitor SIS3. Furthermore, we show that DEX increases cellular ROS levels via upregulation of NADPH oxidase 4 (NOX4) through activation of Smad3, and that SIS3 decreases ROS levels by downregulating NOX4. Instructively, inhibiting NOX4 with GLX351322 and scavenging ROS with NAC were both effective in preventing DEX-induced senescence changes. Similarly, we found in the mouse model that DEX-ac upregulated p15 and NOX4 expression in the trabecular meshwork, with cotreatment with GLX351322 alleviating elevations in IOP. We establish that DEX induces senescence changes in HTMCs by increasing ROS levels via the TGFβ/Smad3/NOX4 axis, increasing IOP and contributing to glaucoma development.

KCNQ1OT1 promotes genome-wide transposon repression by guiding RNA-DNA triplexes and HP1 binding.

Transposon (de)repression and heterochromatin reorganization are dynamically regulated during cell fate determination and are hallmarks of cellular senescence. However, whether they are sequence specifically regulated remains unknown. Here we uncover that the KCNQ1OT1 lncRNA, by sequence-specific Hoogsteen base pairing with double-stranded genomic DNA via its repeat-rich region and binding to the heterochromatin protein HP1α, guides, induces and maintains epigenetic silencing at specific repetitive DNA elements. Repressing KCNQ1OT1 or deleting its repeat-rich region reduces DNA methylation and H3K9me3 on KCNQ1OT1-targeted transposons. Engineering a fusion KCNQ1OT1 with an ectopically targeting guiding triplex sequence induces de novo DNA methylation at the target site. Phenotypically, repressing KCNQ1OT1 induces senescence-associated heterochromatin foci, transposon activation and retrotransposition as well as cellular senescence, demonstrating an essential role of KCNQ1OT1 to safeguard against genome instability and senescence.

Microglia senescence is related to neuropathic pain-associated comorbidities in the spared nerve injury model.

The increased presence of senescent cells in different neurological diseases suggests the contribution of senescence in the pathophysiology of neurodegenerative disorders. Microglia can adapt to any type of disturbance of the homeostasis of the central nervous system, and its altered activity can lead to permanent and unresolvable damage. The aim of this work was to characterize the behavioural phenotype of spared nerve injury mice and then associate it with senescence-related mechanisms. In this work, we investigated the timing of the onset of anxiety, depression, or memory decline associated with peripheral neuropathic pain and their correlation with the presence of microglial cellular senescence. Spared nerve injury mice showed a persistent pain hypersensitivity from 3 days after surgery. Twenty-eight days after nerve injury, they also developed anxiety, depression, and cognitive impairment. The appearance of these symptoms was coincident to a significant increase of senescence markers, such as β-galactosidase and senescent-associated secretory phenotype, at the microglial level in the spinal cord and hippocampus of spared nerve injury animals. These markers were unaltered at previous time points. In murine immortalized microglial cells (BV2) stimulated with LPS 500 ng/mL for 10 days (4 hours/day) every other day, we observed an increase of β-galactosidase and senescent-associated secretory phenotype appearance, a reduction of cell viability, and an increase of senescence-associated heterochromatin foci. Therefore, present findings could represent an important step to a better understanding of the pathophysiological cellular mechanisms in comorbidities related to neuropathic pain states.

Suppression of AGTR1 Induces Cellular Senescence in Hepatocellular Carcinoma Through Inactivating ERK Signaling

Objective: Cellular senescence is an effective barrier against tumorigenesis. Hence, it is of significance to characterize key features of cellular senescence and the induction of senescence in hepatocellular carcinoma (HCC) cells via pharmacological interventions. Our study determined the biological roles as well as mechanisms of angiotensin II type I receptor (AGTR1) on cellular senescence in HCC. Methods: Lentivirus vector-mediated overexpression or knockdown of AGTR1 was conducted in HCC cells, respectively. A volume of 8 μM sorafenib was used to induce cellular senescence, and ERK was activated by 30 ng/ml ERK agonist EGF. Proliferation was evaluated via clone formation assay. HCC cell senescence was examined by flow cytometry for cell cycle, senescence-associated β-galactosidase (SA-β-gal) staining, and senescence-associated heterochromatin foci (SAHF) analysis. AGTR1, p53, p21, extracellular signal-regulated kinase (ERK), and p-ERK expression were assessed through Western blot or immunofluorescence. Results: AGTR1-knockout HCC cells displayed the attenuated proliferative capacity, G2-M phase arrest, increased expression of p53 and p21, and elevated percentages of SA-β-gal- and SAHF-positive cells. In sorafenib-exposed HCC cells, overexpressed AGTR1 enhanced the proliferative capacity and alleviated G2-M phase arrest as well as decreased p53 and p21 expression and the proportions of SA-β-gal- and SAHF-positive cells. Moreover, AGTR1 knockdown attenuated the activity of p-ERK in HCC cells, and ERK agonist ameliorated AGTR1 knockdown-induced cellular senescence. Conclusion: This study demonstrates that suppression of AGTR1 induces cellular senescence in HCC through inactivating ERK signaling. The significant synergistic effect of AGTR1 suppression and sorafenib might represent a potential combination therapy for HCC.

Doxorubicin and SN-38 inhibit the proliferation of osteosarcoma cells by inducing cell cycle arrest

Osteosarcoma is the most common highly malignant primary bone tumor in adolescents and it has a poor prognosis. The aim of the present study was to explore potential chemotherapy drugs for osteosarcoma, in order to provide a theoretical basis for the clinical treatment of the disease. Senescence-associated β-galactosidase (SA-β-gal) staining was used to detect the senescence of drug-treated cells. Bromodeoxyuridine(BRDU) and other methods were used to detect cell proliferation. Immunoprecipitation, immunofluorescence and other techniques were used to explore the impact of drug treatment on the cell cycle. In the present study, doxorubicin (Dox) and 7-ethyl-10-hydroxycamptothecin (SN-38) were found to induce osteosarcoma cell senescence, and senescent cells exhibit the senescence-associated heterochromatin foci phenomenon (SAHF). Cell senescence is induced in osteosarcoma by Dox and SN-38 through inhibiting the expression of the cell cycle-related genes cyclin-dependent kinase (CDK)4, cyclin D1, CDK2 and cyclin E, and inhibit the formation of cell cycle-dependent complexes CDK4/cyclin D1 and CDK2/cyclin E. Eventually, Dox and SN-38 cause cell cycle arrest and induce cell senescence.

Gold and silver plasmonic nanoprobes trace the positions of histone codes

We visualized the distribution of heterochromatin in a single nucleus using plasmonic nanoparticle-conjugated H3K9me3 and H3K27me3 antibodies. Due to distance-dependent plasmonic coupling effects between nanoprobes, their scattering spectra shift to longer wavelengths as the distance between heterochromatin histone markers reduced during oncogene-induced senescence (OIS). These observations were supported by simulating scattering profiles based on considerations of particle numbers, interparticle distances, and the spatial arrangements of plasmonic nanoprobes. Using this plasmon-based colourimetric imaging, we estimated changes in distances between H3K9me3 and H3K27me3 during the formation of senescence-associated heterochromatin foci in OIS cells. We anticipate that the devised analytical technique combined with high-spatial imaging and spectral simulation will eventually lead to a new means of diagnosing and monitoring disease progression and cellular senescence.

Advanced Oxidative Protein Products Drive Trophoblast Cells Into Senescence by Inhibiting the Autophagy: The Potential Implication of Preeclampsia.

Introduction: Advanced oxidation protein products (AOPPs), the novel marker of oxidative stress, have been found to be elevated in preeclampsia (PE). To date, the effect of AOPPs on the senescence of trophoblast cells is still unclear. In this study, we investigated whether AOPPs promoted the senescence of trophoblast cells and explored the underlying mechanisms of AOPPs-induced aging process which may facilitate the progression of PE. Methods: The trophoblast cell line HTR-8/SV neo cells were cultured in the presence of PBS, AOPPs, AOPPs plus an anti-oxidant N-acetyl-L-cysteine (NAC). In some experiments, cells were pre-treated with rapamycin (an activator of autophagy), 3-MA (an inhibitor of autophagy), or cyclic pifithrin-α (PFT-α, an antagonist of p53), and then treated with AOPPs. Cellular senescence was analyzed by measuring the levels of senescence-associated β-galactosidase (SA β-Gal), senescence-associated heterochromatin foci (SAHF), mitochondrial membrane potential (ΔΨm), and cell cycle. Cell autophagic flux was analyzed by measuring tandem fluorescence-tagged LC3 reporter (mCherry-EGFP-LC3). Levels of p53, phosphorylated p53 (p-p53), p21, BECN1, p62, p-mTOR and p-p70S6K were measured by western blot. Results: Treatment with AOPPs significantly increased the levels of SA β-Gal and SAHF, the percentage of cells in the G0/G1 phase, and decreased cell ΔΨm compared with the control group. Co-treatment with NAC and AOPPs significantly reversed AOPPs-induced senescence. Pre-treatment with rapamycin or 3-MA significantly inhibited or promoted AOPPs-induced senescence, respectively. In addition, administration of AOPPs significantly decreased the numbers of mCherry+EGFP+ autophagosomes and mCherry+EGFP- autolysosomes in cells compared with cells treated with PBS. Furthermore, AOPPs significantly increased the levels of proteins p-p53, p21, p-mTOR and p-p70S6K compared with the control group. Pre-treatment with rapamycin or PFT-α significantly down-regulated the levels of SA β-Gal, SAHF, p-p53, p21, autophagy related protein p62, the percentage of cells in the G0/G1 phase, and significantly up-regulated ΔΨm, autophagy related protein BECN1, autophagosomes and autolysosomes compared with cells only treated with AOPPs. Conclusion: AOPPs may induce trophoblast cell senescence by inhibiting the autophagy process in a p53/mTOR/p70S6K-dependent pathway.

Downregulation of JMJD2a and LSD1 is involved in CK2 inhibition-mediated cellular senescence through the p53-SUV39h1 pathway

Lysine methylation is one of the most important histone modifications that modulate chromatin structure. In the present study, the roles of the histone lysine demethylases JMJD2a and LSD1 in CK2 downregulation-mediated senescence were investigated. The ectopic expression of JMJD2a and LSD1 suppressed the induction of senescence-associated β-galactosidase activity and heterochromatin foci formation as well as the reduction of colony-forming and cell migration ability mediated by CK2 knockdown. CK2 downregulation inhibited JMJD2a and LSD1 expression by activating the mammalian target of rapamycin (mTOR)-ribosomal p70 S6 kinase (p70S6K) pathway. In addition, the down-regulation of JMJD2a and LSD1 was involved in activating the p53-p21Cip1/WAF1-SUV39h1-trimethylation of the histone H3 Lys9 (H3K9me3) pathway in CK2-downregulated cells. Further, CK2 downregulation-mediated JMJD2a and LSD1 reduction was found to stimulate the dimethylation of Lys370 on p53 (p53K370me2) and nuclear import of SUV39h1. Therefore, this study indicated that CK2 downregulation reduces JMJD2a and LSD1 expression by activating mTOR, resulting in H3K9me3 induction by increasing the p53K370me2-dependent nuclear import of SUV39h1. These results suggest that CK2 is a potential therapeutic target for age-related diseases.