scholarly journals Μελέτη της κυτταρικής γήρανσης στον ομαλό λειχήνα του στόματος με τη χρήση της ειδικής χρώσης της λιποφουσκίνης με sudan black B, μιά πρωτότυπη μέθοδο ανίχνευσης γηρασμένων κυττάρων

2014 ◽  
Author(s):  
Ελένη Γεωργακοπούλου
Keyword(s):  
Cellular Senescence ◽  
Replicative Senescence ◽  
Sudan Black B ◽  
Beta Galactosidase

Ως κυτταρική γήρανση (cellular senescence) ορίζεται η μη αναστρέψιμη παύση του κυτταρικού κύκλου συνεπεία είτε εξάντλησης των τελομερών, είτε κυτταρικού στρες, και θεωρείται μηχανισμός αντίστασης στην καρκινογένεση. Το φαινόμενο της κυτταρικής γήρανσης αποτελεί μια ερευνητική πρόκληση μιας και αποτελεί συνδετικό κρίκο μεταξύ της φυσιολογικής γήρανσης της χρόνιας φλεγμονής και βασικών μονοπατιών της καρκινογένεσης. Ο μέχρι σήμερα πιο αξιόπιστος δείκτης κυτταρικής γήρανσης είναι η ανίχνευση της δραστηριότητας της β- γαλακτοσιδάσης των γηρασμένων κυττάρων ,(senescence-associated-beta-galactosidase SA-β-gal). Η μέθοδος αυτή δεν μπορεί να εφαρμοστεί σε αρχειακό υλικό (ιστούς εγκιβωτισμένους σε παραφίνη) αλλά μόνο σε φρέσκους ιστούς και σε τομές από άμεσα κατεψυγμένους ιστούς (κρυοτομές). Εξαιτίας αυτού του περιορισμού υπάρχει έλλειψη εκτενών κλινικοπαθολογικών μελετών για την κυτταρική γήρανση.ΣΚΟΠΟΣ : Επιχειρήθηκε η αναζήτηση ενός βιολογικού δείκτη κυτταρικής γήρανσης, με εφαρμογή σε αρχειακό υλικό. Επίσης, μελετήθηκε η πρωτότυπη χρησιμοποίησή του ως δείκτη κυτταρικής γήρανσης σε ένα χρόνιο φλεγμονώδες νόσημα που αποτελεί μοντέλο συσχέτισης χρόνιας φλεγμονής και καρκίνου, τον Ομαλό λειχήνα του στόματος.ΥΛΙΚΑ ΚΑΙ ΜΕΘΟΔΟΙ : Αναζητώντας μια μέθοδο ανίχνευσης γηρασμένων κυττάρων εφαρμόσιμη σε αρχειακό υλικό, αξιολογήσαμε την ιστοχημική χρώση Sudan-Black B (SBB), που είναι ειδική για την ανίχνευση της λιποφουσκίνης. Η λιποφουσκίνη είναι ένα συσσωμάτωμα οξειδωμένων πρωτεϊνών, λιπιδίων και μετάλλων, η οποία συσσωρεύεται σε γηρασμένους ιστούς. Αναλύσαμε κυτταρικά συστήματα στα οποία προκλήθηκε κυτταρική γήρανση είτε ύστερα από εξάντληση του πολλαπλασιασμού (replicative senescence) ή από στρεσογόνα ερεθίσματα, προ-καρκινικές αλλοιώσεις σε υπό προϋποθέσεις knock-in ποντίκια που παρουσιάζουν γήρανση, και τέλος σε ανθρώπινες προκαρκινικές αλλοιώσεις που γνωρίζουμε ότι περιέχουν γηρασμένα κύτταρα. Η τεχνική εν συνεχεία εφαρμόστηκε σε δείγματα Ομαλού λειχήνα, Ακανθοκυτταρικού καρκινώματος στόματος , καλοήθεις βλάβες στοματικού βλεννογόνου (ινώματα) και φυσιολογικού στοματικού βλεννογόνου.ΑΠΟΤΕΛΕΣΜΑΤΑ : Στα παραπάνω πειράματα αποδείξαμε την συνταύτιση της λιποφουσκίνης και του SA-β-gal σε in vitro και in vivo γηρασμένα κύττταρα (κατεψυγμένους ιστούς). Tα ευρήματα αυτά συνηγορούν πως η λιποφουσκίνη είναι ένας ικανός υποψήφιος δείκτης κυτταρικής γήρανσης. Επιπρόσθετα, κατεψυγμένοι ιστοί θετικοί για SA-β-gal μονιμοποιήθηκαν σε φορμόλη, εγκλείστηκαν σε παραφίνη και βάφτηκαν με SBB. Οι αντίστοιχες SA-β-gal θετικές περιοχές στον ιστό βάφτηκαν ειδικά για λιποφουσκίνη με SBB, ενώ οι ιστοί που ήταν αρνητικοί για SA-β-gal βρέθηκαν και αρνητικοί για τη λιποφουσκίνη. Τα ευρήματα αυτά ενισχύουν περαιτέρω την ευαισθησία και την ειδικότητα της SBB χρώσης για την ανάδειξη γηρασμένων κυττάρων σε αρχειακό υλικό. Η τελευταία μοναδική ιδιότητα του SBB μπορεί να αξιοποιηθεί για κλινικοπαθολογικές μελέτες στο ευρέως διαθέσιμο αρχειακό υλικό. Επιπρόσθετα, η εφαρμογή της τεχνικής σε τομές παραφίνης από Ομαλό λειχήνα, Ακανθοκυτταρικό καρκίνωμα σε ινώματα και φυσιολογικό ιστό στόματος, ανέδειξε την παρουσία γηρασμένων ινοβλαστών στις τομές των ινωμάτων και του Ομαλού λειχήνα και την απουσία τους στο φυσιολογικό ιστό και το Ακανθοκυτταρικό καρκίνωμα του στόματος. Τα ευρήματα αυτά συνηγορούν υπερ μιας προστατευτικής δράσης αυτών των κυττάρων , πιθανά στα πλαίσια μιας καλοήθους αντίδρασης του στρώματος.

scholarly journals Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shojiro Katoh ◽  
Atsuki Fujimaru ◽  
Masaru Iwasaki ◽  
Hiroshi Yoshioka ◽  
Rajappa Senthilkumar ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Replicative Senescence ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Human Chondrocytes ◽  
Cartilage Tissue ◽  
Optimal Method ◽  
Beta Galactosidase

AbstractRegenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

Replicative Senescence in Good-Risk Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1922-1922
Author(s):  
Mark Zijlmans ◽  
Susan Swiggers ◽  
Maria Rife Soler ◽  
Berna Beverloo
Keyword(s):  
Cell Growth ◽  
Tumor Cells ◽  
Replicative Senescence ◽  
Proliferative Capacity ◽  
Telomere Shortening ◽  
Normal Cells ◽  
Beta Galactosidase ◽  
Good Risk

Abstract Immortal cell growth is considered the hallmark of tumor cells. In contrast, normal cells have a limited proliferative capacity of 40–60 cell divisions, also known as the Hayflick limit. The limited proliferative capacity of normal cells relates to gradual telomere shortening as a consequence of the end-replication problem. Upon critical telomere shortening, cells enter a non-replicative but viable state referred to as replicative senescence. These replicative senescent cells stain blue in a beta-Galactosidase assay and activate DNA double-strand break repair pathways at telomeres (e.g. gamma-H2AX foci). In human fibroblast models, escape from senescence results from loss of p53 and Rb function. Escape is associated with reactivation of telomerase. High levels of telomerase, as observed in germ cells and most tumor cells, allow for immortal cell growth. Recently, we demonstrated low levels of telomerase in AML patients with t(8;21) or inv(16) (Swiggers et al, G.C.C. 2006). Interestingly, levels of telomerase in these AML samples were similar to levels of telomerase in normal bone marrow progenitor cells. We hypothesized that AML without re-activated telomerase may still have intact senescence pathways that limit the proliferative capacity of normal cells. This hypothesis was addressed by studying AML patient samples without telomerase re-activation, i.e., t(8;21), t(15;17) or inv(16) (n=10), and a control group of AML with telomerase re-activation (multiple gains/losses of genetic material, n=8). AML samples werelong-time cultured in vitro in the presence of hematopoietic growth factors (range 3–6 weeks),analyzed in vivo following transplantation in NOD-SCID mice andin patients at time of relapse. Cells with all characteristics of replicative senescence, i.e. enlarged, viable, non-proliferating, blue-coloring in beta-Galactosidase assay, critical short telomeres and gamma-H2AX foci at telomeres, were clearly observed in all AML samples with t(8;21), t(15;17) or inv(16). Gradual telomere shortening was observed in these AML cells in vitro upon long-term culture, in vivo after transplantation in NOD-SCID mice and in vivo in patients at relapse compared to time of diagnosis, indicating that these AML cells do not have an adequate telomere maintenance mechanism. We conclude that AML cells with t(8;21), t(15;17) or inv(16) are characterized by intact pathways that induce replicative senescence. Intact pathways that limit proliferative lifespan may be critical to the high cure rates following chemotherapy treatment of patients with good-risk AML.

scholarly journals Iron-citrate can induce cell senescent phenotype in human fibroblasts in vitro

2009 ◽  
Vol 15 (S3) ◽  
pp. 15-16
Author(s):  
L. Matos ◽  
H. Almeida
Keyword(s):  
Cell Injury ◽  
Replicative Senescence ◽  
Human Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Senescent Phenotype ◽  
Redox Active ◽  
Stress Induced Premature Senescence ◽  
Beta Galactosidase

AbstractAfter a number of replications, human diploid fibroblasts (HDFs) in culture lose the ability to divide, become insensitive to further proliferation and enter a state of replicative senescence (RS). Subcytotoxic doses of several stressful agents such as hydrogen peroxide, tertbutylhydroperoxide or ethanol, are able to cause stress-induced premature senescence (SIPS) in HDFs in vitro. Such senescent cells display many features of RS as growth arrest, senescence associated beta-galactosidase (SA beta-gal), cell enlargement and overexpression of several genes (e.g., p21, TGF beta-1,IGFBP3). During ageing, iron accumulates in several tissues in vivo, and also in senescent HDFs in vitro. Due to its redox-active properties, it promotes hydroxyl radical production (Fenton reaction) and eventually leads to cell injury. Free radical reactions are known to cause the accumulation of intracellular damage resulting in ageing. Iron may thus be able to cause SIPS. The main objective of the present study was to investigate whether the exposure of HDFs to a subcytotoxic concentration of iron is able to cause SIPS.

scholarly journals Single Cell Transcriptomics Reveals Global Markers of Transcriptional Diversity Across Different Forms of Cellular Senescence

2021 ◽  
Author(s):  
Shane A Evans ◽  
Yee Voan Teo ◽  
Kelly Klark ◽  
Takahiro Ito ◽  
John M Sedivy ◽  
...  
Keyword(s):  
Single Cell ◽  
Cellular Senescence ◽  
Replicative Senescence ◽  
Expression Profiles ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Cell Contact ◽  
Matrix Remodeling

Cellular Senescence is a state of irreversible cell cycle arrest, and the accumulation of senescent cells contributes to agerelated organismal decline. The detrimental effects of cellular senescence are due to the senescence associated secretory phenotype (SASP), an array of signaling molecules and growth factors secreted by senescent cells that contribute to the terile inflammation associated with aging tissues. Recent studies, both in vivo and in vitro, have highlighted the heterogeneous nature of the senescence phenotype. In particular, single cell transcriptomics has revealed that Oncogene Induced Senescence (OIS) is characterized by the presence of subpopulations of cells expressing different SASP profiles. We have generated a comprehensive dataset via single-cell transcriptional profiling of genetically homogenous clonal cell lines from different forms of senescence, including OIS, Replicative Senescence (RS), and DNA Damage Induced Senescence (DDIS). We identified subpopulations of cells that are common to all three major forms of senescence and show that the expression profiles of these subpopulations are driven by markers formerly identified in individual forms of senescence. These common signatures are characterized by chromatin modifiers, inflammation, extracellular matrix remodeling, and Ribosomal protein expression. The expression patterns of these subpopulations recapitulate primary and secondary senescence, a phenomenon where a preexisting (primary) senescent cell induces senescence in a neighboring (secondary) cell through cell-to-cell contact. Since it is still unclear what type of senescence occurs in-vivo with age, it is important to know that the formation of primary and secondary populations is common to multiple types of senescence since this mechanism could help explain how senescent cells accumulate in aged organisms. Finally, we show that these subpopulations show differential susceptibility to the senolytic agent Navitoclax, suggesting that senolytic agents targeting the apoptotic pathways may be clearing only a subset of senescent cells based on their inflammatory profiles in-vivo.

scholarly journals Calcium channel ITPR2 and mitochondria–ER contacts promote cellular senescence and aging

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dorian V. Ziegler ◽  
David Vindrieux ◽  
Delphine Goehrig ◽  
Sara Jaber ◽  
Guillaume Collin ◽  
...  
Keyword(s):  
Cellular Senescence ◽  
Calcium Release ◽  
Liver Steatosis ◽  
Metabolic Stress ◽  
Calcium Release Channel ◽  
Release Channel ◽  
Age Related ◽  
Trisphosphate Receptor

AbstractCellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.

scholarly journals Loss of SATB1 Induces a p21 Dependent Cellular Senescence Phenotype in Dopaminergic Neurons

2018 ◽  
Author(s):  
Markus Riessland ◽  
Benjamin Kolisnyk ◽  
Tae Wan Kim ◽  
Jia Cheng ◽  
Jason Ni ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cellular Senescence ◽  
Dopaminergic Neurons ◽  
Dna Binding Protein ◽  
Dopamine Neurons ◽  
Human Stem Cell ◽  
Transcriptional Program

AbstractCellular senescence is a mechanism used by mitotic cells to prevent uncontrolled cell division. As senescent cells persist in tissues, they cause local inflammation and are harmful to surrounding cells, contributing to aging. Generally, neurodegenerative diseases, such as Parkinson‘s, are disorders of aging. The contribution of cellular senescence to neurodegeneration is still unclear. SATB1 is a DNA binding protein associated with Parkinson’s disease. We report that SATB1 prevents cellular senescence in post-mitotic dopaminergic neurons. Loss of SATB1 causes activation of a cellular senescence transcriptional program in dopamine neurons, both in human stem cell-derived dopaminergic neurons and in mice. We observed phenotypes which are central to cellular senescence in SATB1 knockout dopamine neurons in vitro and in vivo. Moreover, we found that SATB1 directly represses expression of the pro-senescence factor, p21, in dopaminergic neurons. Our data implicate senescence of dopamine neurons as a contributing factor to the pathology of Parkinson’s disease.

scholarly journals Exploring the effect of nsSNPs in human YPEL3 gene in cellular senescence

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Abhishek Singh ◽  
Mukesh Thakur ◽  
Sujeet Kumar Singh ◽  
Lalit Kumar Sharma ◽  
Kailash Chandra
Keyword(s):  
Conformational Changes ◽  
Cellular Senescence ◽  
Health Professionals ◽  
Md Simulation ◽  
Tumour Cells ◽  
In Vivo Studies ◽  
Native Conformation ◽  
Altered Expression

Abstract YPEL3 that induces cellular senescence in both normal and tumour cells of humans may show altered expression under the influence of incidental mutations. In this study, we proposed the first structure of Native YPEL3 protein and its five possible deleterious mutants—V40M, C61Y, G98R, G108S, and A131T and predicted their deleterious effects to alter stability, flexibility and conformational changes in the protein. The MD simulation (RMSD, RMSF, Rg, h-bond and SASA) analysis revealed that the variants V40M, G98R and G108S increased the flexibility in protein, and variant V40M imparted more compactness to the protein.. In general, variants attributed changes in the native conformation and structure of the YPEL3 protein which might affect the native function of cellular senescence. The study provides opportunities for health professionals and practitioners in formulating précised medicines to effectively cure various cancers. We propose in-vitro or in-vivo studies should consider these reported nsSNPs while examining any malfunction in the YPEL3 protein.

scholarly journals P01.16 Effects of the STAT3 inhibitors on senescent tumour cells

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A16.1-A16
Author(s):  
O Sapega ◽  
R Mikyskova ◽  
K Musilek ◽  
J Bieblova ◽  
Z Hodny ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Czech Republic ◽  
Cell Lines ◽  
Cellular Senescence ◽  
Tumour Cells ◽  
Stat3 Signaling ◽  
Stat3 Signaling Pathway ◽  
Stat3 Phosphorylation

BackgroundCellular senescence is the process of cell proliferation arrest. Premature cellular senescence can be induced by chemotherapy, irradiation and, under certain circumstances, by cytokines. Senescent cells produce a number of secreted proteins and growth factors that may either stimulate or inhibit cell proliferation. One of the major cytokines that play role in regulation of cellular senescence is IL-6. IL-6/STAT3 signaling pathway represent decisive regulatory factors in cellular senescence. The objective of this study was to compare the effects of the STAT3 inhibitors on senescent and proliferative tumour cells. Further, the therapeutic potential of the STAT3 inhibitors was evaluated using murine tumour models.Materials and MethodsIn vitro, as well as in vivo experiments were performed using TC-1 (model for HPV16-associated tumours) TRAMP-C2 (prostate cancer) cell lines. C57Bl/6NCrl mice, 7–8 weeks old, were obtained from Velaz (Prague, Czech Republic). Experimental protocols were approved by the Institutional Animal Care Committee of the Institute of Molecular Genetics (Prague, Czech Republic). STAT3 inhibitors, namely STATTIC, BP-102 (synthesised at the University of Hradec Kralove) and their derivatives were tested for their effects on tumour cells, such as cytotoxicity, ability to inhibit STAT3 phosphorylation, cell proliferation and tumour growth in syngeneic mice.ResultsWe have previously demonstrated that docetaxel-induced senescence in the TC-1 and TRAMP-C2 murine tumour cell lines, which was proved by in vitro (detection of increased p21 expression, positive beta-galactosidase staining, and the typical SASP capable to induce ‘bystander’ senescence), and in vivo experiments, using C57BL/6 mice [1]. Both TC-1 and TRAMP-C2 cells displayed elevated IL-6 secretion and activated STAT3 signaling pathway. Therefore, we tested efficacy of the STAT3 inhibitors on these cell lines. Cytotoxic and STAT3 phosphorylation inhibitory effects of the inhibitors were observed in both proliferating and senescent cells. Antitumor effects of selected inhibitors were evaluated.ConclusionsCollectively, STAT3 is an attractive target for therapeutic approaches in cancer treatment and we can assume that inhibition of the STAT3 pathway can be used for elimination of the pernicious effects of the senescent cells.ReferenceSimova J, Sapega O, Imrichova T, Stepanek I, Kyjacova L, Mikyskova R, Indrova M, Bieblova J, Bubenik J, Bartek J, et al: Tumor growth accelerated by chemotherapy-induced senescent cells is suppressed by treatment with IL-12 producing cellular vaccines. Oncotarget7: 54952–54964, 2016. This work was supported by the research grant No. NV18-05-00562 provided by the Grant Agency of the Ministry of Health of the Czech Republic.Disclosure InformationO. Sapega: None. R. Mikyskova: None. K. Musilek: None. J. Bieblova: None. Z. Hodny: None. M. Reinis: None.

scholarly journals Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV

2007 ◽  
Vol 27 (8) ◽  
pp. 3123-3130 ◽  
Author(s):  
Klaus Fortschegger ◽  
Bettina Wagner ◽  
Regina Voglauer ◽  
Hermann Katinger ◽  
Maria Sibilia ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Replicative Senescence ◽  
Inner Cell Mass ◽  
Umbilical Vein ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Proliferative Potential ◽  
Mouse Development

ABSTRACT SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

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