scholarly journals Interphase Chromosomes in Replicative Senescence: Chromosome Positioning as a Senescence Biomarker and the Lack of Nuclear Motor-Driven Chromosome Repositioning in Senescent Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Ishita S. Mehta ◽  
Kumars Riyahi ◽  
Rita Torres Pereira ◽  
Karen J. Meaburn ◽  
Martin Figgitt ◽  
...  
Keyword(s):  
Heat Shock ◽  
Replicative Senescence ◽  
Culture Media ◽  
Nuclear Periphery ◽  
Chromosome Territory ◽  
Dermal Fibroblasts ◽  
Serum Starvation ◽  
Chromosome Movement ◽  
Protein Activity ◽  
Quiescent Cells

This study demonstrates, and confirms, that chromosome territory positioning is altered in primary senescent human dermal fibroblasts (HDFs). The chromosome territory positioning pattern is very similar to that found in HDFs made quiescent either by serum starvation or confluence; but not completely. A few chromosomes are found in different locations. One chromosome in particular stands out, chromosome 10, which is located in an intermediate location in young proliferating HDFs, but is found at the nuclear periphery in quiescent cells and in an opposing location of the nuclear interior in senescent HDFs. We have previously demonstrated that individual chromosome territories can be actively and rapidly relocated, with 15 min, after removal of serum from the culture media. These chromosome relocations require nuclear motor activity through the presence of nuclear myosin 1β (NM1β). We now also demonstrate rapid chromosome movement in HDFs after heat-shock at 42°C. Others have shown that heat shock genes are actively relocated using nuclear motor protein activity via actin or NM1β (Khanna et al., 2014; Pradhan et al., 2020). However, this current study reveals, that in senescent HDFs, chromosomes can no longer be relocated to expected nuclear locations upon these two types of stimuli. This coincides with a entirely different organisation and distribution of NM1β within senescent HDFs.

scholarly journals The dark-side of the outside: how extracellular heat shock proteins promote cancer

2021 ◽  
Author(s):  
Laura Seclì ◽  
Federica Fusella ◽  
Lidia Avalle ◽  
Mara Brancaccio
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Epithelial To Mesenchymal Transition ◽  
Dark Side ◽  
Migration And Invasion ◽  
Protein Activity ◽  
Tumour Immunity ◽  
Mesenchymal Transition ◽  
Cancer Hallmarks ◽  
Shock Proteins

AbstractIn addition to exerting several essential house-keeping activities in the cell, heat shock proteins (HSPs) are crucial players in a well-structured molecular program activated in response to stressful challenges. Among the different activities carried out by HSPs during emergency, they reach the extracellular milieu, from where they scout the surroundings, regulate extracellular protein activity and send autocrine and paracrine signals. Cancer cells permanently experience stress conditions due to their altered equilibrium and behaviour, and constantly secrete heat shock proteins as a result. Other than supporting anti-tumour immunity, extracellular heat shock proteins (eHSPs), can also exacerbate cancer cell growth and malignancy by sustaining different cancer hallmarks. eHSPs are implicated in extracellular matrix remodelling, resistance to apoptosis, promotion of cell migration and invasion, induction of epithelial to mesenchymal transition, angiogenesis and activation of stromal cells, supporting ultimately, metastasis dissemination. A broader understanding of eHSP activity and contribution to tumour development and progression is leading to new opportunities in the diagnosis and treatment of cancer.

scholarly journals Heterogeneous chromatin mobility derived from chromatin states is a determinant of genome organisation in S. cerevisiae

2017 ◽  
Author(s):  
Sven A. Sewitz ◽  
Zahra Fahmi ◽  
Latifa Aljebali ◽  
Jeremy Bancroft ◽  
Otávio J. B. Brustolini ◽  
...  
Keyword(s):  
Heat Shock ◽  
Spatial Clustering ◽  
Genome Structure ◽  
Nuclear Periphery ◽  
Three Dimensional ◽  
Normal Growth ◽  
Heterogeneous Distribution ◽  
Chromatin States ◽  
Protein Occupancy ◽  
Self Organisation

AbstractSpatial organisation of the genome is essential for regulating gene activity, yet the mechanisms that shape this three-dimensional organisation in eukaryotes are far from understood. Here, we combine bioinformatic determination of chromatin states during normal growth and heat shock, and computational polymer modelling of genome structure, with quantitative microscopy and Hi-C to demonstrate that differential mobility of yeast chromosome segments leads to spatial self-organisation of the genome. We observe that more than forty percent of chromatin-associated proteins display a poised and heterogeneous distribution along the chromosome, creating a heteropolymer. This distribution changes upon heat shock in a concerted, state-specific manner. Simulating yeast chromosomes as heteropolymers, in which the mobility of each segment depends on its cumulative protein occupancy, results in functionally relevant structures, which match our experimental data. This thermodynamically driven self-organisation achieves spatial clustering of poised genes and mechanistically contributes to the directed relocalisation of active genes to the nuclear periphery upon heat shock.One Sentence SummaryUnequal protein occupancy and chromosome segment mobility drive 3D organisation of the genome.

scholarly journals The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis

2021 ◽  
Vol 22 (23) ◽  
pp. 12827
Author(s):  
Mahshid Ghasemi ◽  
Tyron Turnbull ◽  
Sonia Sebastian ◽  
Ivan Kempson
Keyword(s):  
Cell Viability ◽  
Single Cell ◽  
Metabolic Activity ◽  
Mtt Assay ◽  
Single Cell Analysis ◽  
Culture Media ◽  
Cancer Cell Line ◽  
Prostate Cancer Cell Line ◽  
Serum Starvation ◽  
Treatment Toxicity

The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations.

scholarly journals Heat shock protein 27 promotes cell cycle progression by down-regulating E2F transcription factor 4 and retinoblastoma family protein p130

2018 ◽  
Vol 293 (41) ◽  
pp. 15815-15826 ◽  
Author(s):  
Ah-Mee Park ◽  
Ikuo Tsunoda ◽  
Osamu Yoshie
Keyword(s):  
Cell Cycle ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Cellular Senescence ◽  
Cell Cycle Progression ◽  
Heat Shock Protein 27 ◽  
Serum Starvation ◽  
Cell Cycle Gene ◽  
Cycle Progression ◽  
Human Lung Fibroblast

Heat shock protein 27 (HSP27) protects cells under stress. Here, we demonstrate that HSP27 also promotes cell cycle progression of MRC-5 human lung fibroblast cells. Serum starvation for 24 h induced G1 arrest in these cells, and upon serum refeeding, the cells initiated cell cycle progression accompanied by an increase in HSP27 protein levels. HSP27 levels peaked at 12 h, and transcriptional up-regulation of six G2/M-related genes (CCNA2, CCNB1, CCNB2, CDC25C, CDCA3, and CDK1) peaked at 24–48 h. siRNA-mediated HSP27 silencing in proliferating MRC-5 cells induced G2 arrest coinciding with down-regulation of these six genes. Of note, the promoters of all of these genes have the cell cycle–dependent element and/or the cell cycle gene-homology region. These promoter regions are known to be bound by the E2F family proteins (E2F-1 to E2F-8) and retinoblastoma (RB) family proteins (RB1, p107, and p130), among which E2F-4 and p130 were strongly up-regulated in HSP27-knockdown cells. E2F-4 or p130 knockdown concomitant with the HSP27 knockdown rescued MRC-5 cells from G2 arrest and up-regulated the six cell cycle genes. Moreover, we observed cellular senescence in MRC-5 cells on day 3 after the HSP27 knockdown, as evidenced by increased senescence-associated β-gal activity and up-regulated inflammatory cytokines. The cellular senescence was also suppressed by the concomitant knockdown of E2F-4/HSP27 or p130/HSP27. Our findings indicate that HSP27 promotes cell cycle progression of MRC-5 cells by suppressing expression of the transcriptional repressors E2F-4 and p130.

Abstract 132: A Novel Hydrogen Sulfide (H 2 S) Prodrug, SG1002, Protects Against Myocardial Oxidative Damage and Hypertrophic Signaling via Induction of Cystathionine Β-Synthase (CBS) and Antioxidant Proteins

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Kazi N Islam ◽  
Erminia Donnarumma ◽  
Erinn Donnelly ◽  
David J Lefer
Keyword(s):  
Oxidative Stress ◽  
Clinical Utility ◽  
Culture Media ◽  
Therapeutic Strategies ◽  
Oxidation Products ◽  
Cellular Damage ◽  
Serum Starvation ◽  
Antioxidant Proteins ◽  
Hypertrophic Signaling ◽  
Cells Cultured

Background: Endogenously produced H 2 S is critical for cardiovascular homeostasis. Therapeutic strategies aimed at increasing H 2 S levels have proven cardioprotective in models of acute myocardial infarction and heart failure (HF). The present study was under taken to investigate the effects of a novel H 2 S prodrug, SG1002, on stress induced hypertrophic signaling in murine HL1 cardiomyocytes. Methods: HL1 cells were maintained either in serum starvation (1%) or serum containing (10%) media followed by treatment either with SG1002 or H 2 O 2 , or endothelin-1 (ET-1)/phenylephrine (Phe) or in combination. Treated cells were analyzed for specific experimental needs. Results: SG1002 significantly increased cellular levels of the H 2 S producing enzyme, CBS, as well as production of H 2 S and nitrosothiol in HL1 cells cultured both in serum starvation or serum containing media. SG1002 significantly inhibited H 2 O 2 and ET-1/Phe induced oxidative stress in both culture media as measured by advanced protein oxidation products and MDA levels. Expression of ANP and BNP were markedly attenuated by SG1002 treatment. Cells cultured in media supplemented with serum containing H 2 O 2 /(ET-1 or Phe) or in 1% serum exhibited decreased levels of CBS, SOD1, and catalase. When the HL1 cells were coincubated with SG1002 cellular damage from oxidative stress was significantly attenuated accompanied by an increase in CBS expression. Conclusion: Our data clearly demonstrate that SG1002 attenuates myocardial cellular damage via increasing antioxidant proteins. SG1002 directly increases H 2 S levels and upregulates CBS. Studies are currently underway to evaluate the clinical utility of SG1002 in HF.

Photobiomodulation with 590 nm Wavelength Delays the Telomere Shortening and Replicative Senescence of Human Dermal Fibroblasts In Vitro

2020 ◽  
Vol 38 (11) ◽  
pp. 656-660
Author(s):  
Borislav Arabadjiev ◽  
Roumen Pankov ◽  
Ivelina Vassileva ◽  
Lyuben Sashov Petrov ◽  
Ivan Buchvarov
Keyword(s):  
Replicative Senescence ◽  
Dermal Fibroblasts ◽  
Human Dermal Fibroblasts ◽  
Telomere Shortening

311 EFFECT OF HEAT SHOCK DURING THE FERTILIZATION AND CULTURE PERIOD ON BOVINE EMBRYO DEVELOPMENT AND THE PROTECTIVE EFFECT OF BROWN ALGAE PHLOROTANNINS

2007 ◽  
Vol 19 (1) ◽  
pp. 271
Author(s):  
M. Sakatani ◽  
K. Nagayama ◽  
K. Kobayashi ◽  
K. Kobayashi ◽  
K. Morishita ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protective Effect ◽  
Embryo Development ◽  
Culture Media ◽  
Reproductive Function ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Mean Values ◽  
Bovine Sperm ◽  
Intracellular Ros

It is widely reported that heat stress adversely affects the reproductive function of cattle, such as ovarian functions, fertilization, and embryo development. In a previous study, we reported that heat shock decreases embryo development and increases intracellular reactive oxygen species (ROS). Also some antioxidants increase embryo development under conditions of heat shock by reducing the intracellular ROS. Phlorotannins extracted from brown alga are known as a strong antioxidant. However, heat shock and the antioxidative effect of phlorotannins on fertilization and embryo development has not been carefully studied. In the present study, we investigated the effect of heat shock on fertilization and early embryo development, and the protective effect of phlorotannins on embryo development under conditions of heat shock. In all experiments, bovine oocytes were collected from the local abattoir and matured with TCM-199 (Experiment 1). Bovine sperm drops prepared by BO solution were pretreated at 41�C for 4 h with or without 100 ng mL-1 of phlorotannins. After heat shock, oocytes were fertilized in drops at 38.5�C for 6 h. Putative zygotes were cultured with CR1 + 5% FCS at 38.5�C. The percentages of embryos cleaved and developed to blastocysts were evaluated on Days 2 and 8. The percentages of embryo division and development were compared with embryos fertilized with sperm pretreated at 38.5�C for 4 h (Experiment 2). Oocytes were fertilized at 41�C for 6 h with or without 100 ng mL-1 of phlorotannins. Putative zygotes were cultured with CR1 + 5% FCS at 38.5�C. On Days 2 and 8, the percentages of cleaved embryos and those developed to blastocysts were evaluated and compared with embryos fertilized at 38.5�C for 6 h (Experiment 3). Oocytes were fertilized at 38.5�C for 6 h. Putative zygotes were cultured with or without 10 ng mL-1 of phlorotannins in CR1 + 5% FCS. On Day 2, embryos were exposed to 41�C for 6 h as heat shock. After heat shock, embryos were cultured at 38.5�C to Day 8, and embryo development was evaluated. The percentages of embryo development were compared with those for embryos cultured at 38.5�C through to Day 8 without phlorotannins. Mean values were compared by Student's t-test. There were no significant differences in the percentages of embryo cleavage among all experiments. The percentages of embryo development were significantly (P < 0.05) decreased by heat shock in all experiments [Experiment 1: 45.0 vs. 29.2%; Experiment 2: 25.1 vs. 6.6%; Experiment 3: 28.6 vs. 15.3% (control vs. heat shock)]. In contrast, the addition of phlorotannins to the fertilization or culture media tended to improve the embryo development (Experiment 1: 41.9%; Experiment 2: 15.1%; Experiment 3: 22.2%). These results indicate that heat shock affects not only embryo development but also fertilization. And under conditions of heat shock, the addition of phlorotannins would be effective in improving embryo development from fertilization to development.

Calcium homeostasis in cardiomyocytes isolated from heat-shocked rats

1996 ◽  
Vol 271 (5) ◽  
pp. H1938-H1946 ◽  
Author(s):  
R. N. Cornelussen ◽  
L. Ver Donck ◽  
G. Verellen ◽  
M. Borgers ◽  
G. J. van der Vusse ◽  
...  
Keyword(s):  
Heat Stress ◽  
Heat Shock ◽  
Simultaneous Measurement ◽  
Left Ventricular ◽  
Cellular Mechanism ◽  
Fluorescence Ratio ◽  
Adult Rats ◽  
Quiescent Cells ◽  
Contractile Performance

The cellular mechanism of heat shock-mediated cardioprotection is still under debate. Because heat pretreatment negatively affects the normoxic left ventricular contractile performance in vitro when the extracellular Ca2+ concentration ([Ca2+]o) is relatively low (0.65-1.25 mM), the intracellular Ca2+ homeostasis was studied in more detail in cardiomyocytes isolated from adult rats 24 h after heat stress (42 degrees C for 15 min) or anesthesia (control). Sensitivity to Ca2+ overload was assessed by exposure to veratridine (quiescent cells) or to [Ca2+]o ranging from 0.125 to 20 mM in quiescent and paced cardiomyocytes. The fraction of irreversibly hypercontracted cells was not different between groups. The fura-2 fluorescence ratio (I340/I380), which was used as a measure for cytoplasmic Ca2+ concentration ([Ca2+]i) in quiescent cells after exposure to [Ca2+]o (0.5-10 mM), was also not different between groups. Myofilament Ca2+ sensitivity was assessed in paced (0.5 Hz) cells by simultaneous measurement of [Ca2+]i transients and cell shortening. At stepwise increases of [Ca2+]o from 1 to 10 mM, these parameters were comparable between groups. The diastolic cell length shortened progressively and equally in both groups after increasing [Ca2+]o. However, within 2 min of return from 10 to 1 mM [Ca2+]o, cells from heat-shocked rats retained the same length, whereas cells from control rats contracted further (P = 0.05). These data suggest that heat stress improves relaxation after challenge with high [Ca2+]o.

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