Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
Am J Hum Genet ; 105(3): 493-508, 2019 09 05.
Article in English | MEDLINE | ID: mdl-31447100

ABSTRACT

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.


Subject(s)
Cellular Senescence/physiology , Histones/physiology , Aneuploidy , Cell Nucleolus/metabolism , Child , Chromatin/metabolism , DNA Methylation , Female , Histones/chemistry , Humans , Infant , Male , Middle Aged
2.
Cells ; 8(7)2019 07 12.
Article in English | MEDLINE | ID: mdl-31336873

ABSTRACT

Cancer cells need to acquire telomere maintenance mechanisms in order to counteract progressive telomere shortening due to multiple rounds of replication. Most human tumors maintain their telomeres expressing telomerase whereas the remaining 15%-20% utilize the alternative lengthening of telomeres (ALT) pathway. Previous studies have demonstrated that ionizing radiations (IR) are able to modulate telomere lengths and to transiently induce some of the ALT-pathway hallmarks in normal primary fibroblasts. In the present study, we investigated the telomere length modulation kinetics, telomeric DNA damage induction, and the principal hallmarks of ALT over a period of 13 days in X-ray-exposed primary cells. Our results show that X-ray-treated cells primarily display telomere shortening and telomeric damage caused by persistent IR-induced oxidative stress. After initial telomere erosion, we observed a telomere elongation that was associated to the transient activation of a homologous recombination (HR) based mechanism, sharing several features with the ALT pathway observed in cancer cells. Data indicate that telomeric damage activates telomeric HR-mediated repair in primary cells. The characterization of HR-mediated telomere repair in normal cells may contribute to the understanding of the ALT pathway and to the identification of novel strategies in the treatment of ALT-positive cancers.


Subject(s)
DNA Repair/physiology , DNA/metabolism , Fibroblasts , Telomere Homeostasis , Telomere Shortening , Telomere/metabolism , Cell Line , DNA Damage , Fibroblasts/cytology , Fibroblasts/metabolism , Homologous Recombination , Humans , X-Rays
3.
Environ Mol Mutagen ; 60(7): 568-575, 2019 08.
Article in English | MEDLINE | ID: mdl-30942920

ABSTRACT

The effects of ultrasound on the cytoskeleton, comprising microtubules, had been studied decades ago. Nonetheless, very little attention has been paid to the effects of ultrasound on the mitotic spindle, which is also formed by microtubules. In this study, we treated human fibroblasts and human cancer cells (HeLa and MCF-7) with 1-MHz ultrasound at low intensities (70, 140, and 300 mW/cm2 ). In all cell lines, 5 min after the end of sonication, we found an intensity-dependent increase of mitotic abnormalities (including multipolar spindles). Two hours after sonication, these abnormalities were present, but at much lower frequencies. Twenty-four hours after sonication, mitotic abnormalities were at the same level of untreated samples, suggesting a transient effect due to ultrasound. Beside abnormalities of the mitotic spindle, we also observed an increase of metaphases with nonaligned chromosomes. The mitotic index of fibroblasts and HeLa cells, two hours after sonication, showed an intensity-dependent decrease; this was not observed in MCF-7 cells. In agreement with this last result, ultrasound-induced growth inhibition (which was also intensity-dependent) was more marked in fibroblasts and HeLa cells compared to MCF-7 cells. This work indicates that therapeutic ultrasound, even at intensities below the cavitation threshold, can affect genome integrity, showing the need to increase the knowledge of the potential risks of ultrasound to human health. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.


Subject(s)
Spindle Apparatus/physiology , Cell Line , Cell Line, Tumor , Chromosomes/physiology , Fibroblasts/physiology , HeLa Cells , Humans , MCF-7 Cells , Mitosis/physiology , Sonication/methods
4.
Cells ; 8(1)2019 01 03.
Article in English | MEDLINE | ID: mdl-30609792

ABSTRACT

Oxidative DNA damage, particularly 8-oxoguanine, represents the most frequent DNA damage in human cells, especially at the telomeric level. The presence of oxidative lesions in the DNA can hinder the replication fork and is able to activate the DNA damage response. In this study, we wanted to understand the mechanisms by which oxidative damage causes telomere dysfunction and senescence in human primary fibroblasts. After acute oxidative stress at telomeres, our data demonstrated a reduction in TRF1 and TRF2, which are involved in proper telomere replication and T-loop formation, respectively. Furthermore, we observed a higher level of γH2AX with respect to 53BP1 at telomeres, suggesting a telomeric replication fork stall rather than double-strand breaks. To confirm this finding, we studied the replication of telomeres by Chromosome Orientation-FISH (CO-FISH). The data obtained show an increase in unreplicated telomeres after hydrogen peroxide treatment, corroborating the idea that the presence of 8-oxoG can induce replication fork arrest at telomeres. Lastly, we analyzed the H3K9me3 histone mark after oxidative stress at telomeres, and our results showed an increase of this marker, most likely inducing the heterochromatinization of telomeres. These results suggest that 8-oxoG is fundamental in oxidative stress-induced telomeric damage, principally causing replication fork arrest.


Subject(s)
Cellular Senescence/genetics , DNA Replication , Oxidative Stress/genetics , Telomere/genetics , Cells, Cultured , DNA Damage , Fibroblasts/cytology , Guanine/analogs & derivatives , Guanine/pharmacology , Histones/metabolism , Humans , Hydrogen Peroxide/pharmacology , Primary Cell Culture , Shelterin Complex , Telomere-Binding Proteins/metabolism , Telomeric Repeat Binding Protein 2/metabolism , Tumor Suppressor p53-Binding Protein 1/metabolism
5.
EMBO Rep ; 20(2)2019 02.
Article in English | MEDLINE | ID: mdl-30591524

ABSTRACT

Telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) constitute the core telomerase enzyme that maintains the length of telomeres. Telomere maintenance is affected in a broad range of cancer and degenerative disorders. Taking advantage of gain- and loss-of-function approaches, we show that Argonaute 2 (AGO2) promotes telomerase activity and stimulates the association between TERT and TERC AGO2 depletion results in shorter telomeres as well as in lower proliferation rates in vitro and in vivo We also demonstrate that AGO2 interacts with TERC and with a newly identified sRNA (terc-sRNA), arising from the H/ACA box of TERC Notably, terc-sRNA is sufficient to enhance telomerase activity when overexpressed. Analyses of sRNA-Seq datasets show that terc-sRNA is detected in primary human tissues and increases in tumors as compared to control tissues. Collectively, these data uncover a new layer of complexity in the regulation of telomerase activity by AGO2 and might lay the foundation for new therapeutic targets in tumors and telomere diseases.


Subject(s)
Argonaute Proteins/metabolism , RNA/genetics , RNA/metabolism , Telomerase/metabolism , Animals , Argonaute Proteins/genetics , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Enzyme Activation , Gene Expression , Genetic Loci , Heterografts , Humans , Mice , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Nucleic Acid Conformation , Protein Binding , RNA/chemistry , Telomerase/chemistry , Telomerase/genetics
6.
FEBS J ; 285(20): 3769-3785, 2018 10.
Article in English | MEDLINE | ID: mdl-30095224

ABSTRACT

In the present paper, the biological effects of three different naphthalene diimides (NDIs) G-quadruplex (G4) ligands (H-NDI-Tyr, H-NDI-NMe2, and tetra-NDI-NMe2) were comparatively evaluated to those exerted by RHPS4, a well-characterized telomeric G4-ligand, in an in vitro model of glioblastoma. Data indicated that NDIs were very effective in blocking cell proliferation at nanomolar concentrations, although displaying a lower specificity for telomere targeting compared to RHPS4. In addition, differently from RHPS4, NDIs failed to enhance the effect of ionizing radiation, thus suggesting that additional targets other than telomeres could be involved in the strong NDI-mediated anti-proliferative effects. In order to test telomeric off-target action of NDIs, a panel of genes involved in tumor progression, DNA repair, telomere maintenance, and cell-cycle regulation were evaluated at transcriptional and translational level. Specifically, the compounds were able to cause a marked reduction of TERT and BCL2 amounts as well as to favor the accumulation of proteins involved in cell cycle control. A detailed cytofluorimetric analysis of cell cycle progression by means of bromodeoxyuridine (BrdU) incorporation and staining of phospho-histone H3 indicated that NDIs greatly reduce the progression through S-phase and lead to G1 accumulation of BrdU-positive cells. Taken together, these data indicated that, besides effects on telomeres and oncogenes such as Tert and Bcl2, nanomolar concentrations of NDIs determined a sustained block of cell proliferation by slowing down cell cycle progression during S-phase. In conclusion, our data indicate that NDIs G4-ligands are powerful antiproliferative agents, which act through mechanisms that ultimately lead to altered cell-cycle control.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle/drug effects , Cell Proliferation/drug effects , G-Quadruplexes , Gene Expression Regulation, Neoplastic/drug effects , Glioma/pathology , Imides/chemistry , Naphthalenes/chemistry , Telomere , Acridines/pharmacology , Antineoplastic Agents/chemistry , Glioma/drug therapy , Glioma/genetics , Histones/genetics , Histones/metabolism , Humans , Ligands , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Telomerase/genetics , Telomerase/metabolism , Tumor Cells, Cultured
7.
Health Phys ; 115(1): 126-139, 2018 07.
Article in English | MEDLINE | ID: mdl-29787439

ABSTRACT

In the last decades, technological development has led to an increasing use of devices and systems based on microwave radiation. The increased employment of these devices has elicited questions about the potential long-term health consequences associated with microwave radiation exposure. From this perspective, biological effects of microwave radiation have been the focus of many studies, but the reported scientific data are unclear and contradictory. The aim of this study is to evaluate the potential genotoxic and cellular effects associated with in vitro exposure of human fetal and adult fibroblasts to microwave radiation at the frequency of 25 GHz. For this purpose, several genetic and biological end points were evaluated. Results obtained from comet assay, phosphorylation of H2AX histone, and antikinetochore antibody (CREST)-negative micronuclei frequency excluded direct DNA damage to human fetal and adult fibroblasts exposed to microwaves. No induction of apoptosis or changes in prosurvival signalling proteins were detected. Moreover, CREST analysis showed for both the cell lines an increase in the total number of micronuclei and centromere positive micronuclei in exposed samples, indicating aneuploidy induction due to chromosome loss.


Subject(s)
Fetus/pathology , Fibroblasts/pathology , Micronuclei, Chromosome-Defective/radiation effects , Microwaves/adverse effects , Adult , Aneuploidy , Cells, Cultured , Comet Assay , DNA Damage/radiation effects , Fetus/radiation effects , Fibroblasts/radiation effects , Histones/genetics , Humans , Micronucleus Tests
8.
Environ Mol Mutagen ; 59(6): 476-487, 2018 07.
Article in English | MEDLINE | ID: mdl-29602275

ABSTRACT

The applications of Terahertz (THz) technologies have significantly developed in recent years, and the complete understanding of the biological effects of exposure to THz radiation is becoming increasingly important. In a previous study, we found that THz radiation induced genomic damage in fetal fibroblasts. Although these cells demonstrated to be a useful model, exposure of human foetuses to THz radiation is highly improbable. Conversely, THz irradiation of adult dermal tissues is cause of possible concern for some professional and nonprofessional categories. Therefore, we extended our study to the investigation of the effects of THz radiation on adult fibroblasts (HDF). In this work, the effects of THz exposure on HDF cells genome integrity, cell cycle, cytological ultrastructure and proteins expression were assessed. Results of centromere-negative micronuclei frequencies, phosphorylation of H2AX histone, and telomere length modulation indicated no induction of DNA damage. Concordantly, no changes in the expression of proteins associated with DNA damage sensing and repair were detected. Conversely, our results showed an increase of centromere-positive micronuclei frequencies and chromosomal nondisjunction events, indicating induction of aneuploidy. Therefore, our results indicate that THz radiation exposure may affect genome integrity through aneugenic effects, and not by DNA breakage. Our findings are compared to published studies, and possible biophysical mechanisms are discussed. Environ. Mol. Mutagen. 59:476-487, 2018. © 2018 Wiley Periodicals, Inc.


Subject(s)
Aneuploidy , Chromosome Aberrations/radiation effects , Fibroblasts/radiation effects , Terahertz Radiation/adverse effects , Adult , Cell Cycle/radiation effects , Cell Line , DNA Damage/radiation effects , Fibroblasts/cytology , Fibroblasts/metabolism , Genomic Instability/radiation effects , Humans , Micronucleus Tests , Telomere Homeostasis/radiation effects
9.
Stem Cell Res Ther ; 8(1): 271, 2017 Nov 28.
Article in English | MEDLINE | ID: mdl-29183402

ABSTRACT

BACKGROUND: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. METHODS: Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. RESULTS: Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm-1, which is enriched in human induced pluripotent stem cells. CONCLUSIONS: Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.


Subject(s)
DNA/genetics , Human Embryonic Stem Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , RNA/genetics , Spectrum Analysis, Raman , Biomarkers/metabolism , Cell Cycle/genetics , Cell Differentiation , Cluster Analysis , DNA/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , Gene Expression Profiling , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Human Embryonic Stem Cells/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Karyotyping , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Primary Cell Culture , Principal Component Analysis , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Sendai virus/genetics , Sendai virus/metabolism , Transfection
10.
Sci Rep ; 7: 43309, 2017 02 27.
Article in English | MEDLINE | ID: mdl-28240303

ABSTRACT

Cells are often subjected to the effect of reactive oxygen species (ROS) as a result of both intracellular metabolism and exposure to exogenous factors. ROS-dependent oxidative stress can induce 8-oxodG within the GGG triplet found in the G-rich human telomeric sequence (TTAGGG), making telomeres highly susceptible to ROS-induced oxidative damage. Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes and their dysfunction is believed to affect a wide range of cellular and/or organismal processes. Acute oxidative stress was shown to affect telomere integrity, but how prolonged low level oxidative stress, which may be more physiologically relevant, affects telomeres is still poorly investigated. Here, we explored this issue by chronically exposing human primary fibroblasts to a low dose of hydrogen peroxide. We observed fluctuating changes in telomere length and fluctuations in the rates of chromosome instability phenotypes, such that when telomeres shortened, chromosome instability increased and when telomeres lengthened, chromosome instability decreased. We found that telomere length fluctuation is associated with transient activation of an alternative lengthening of telomere (ALT) pathway, but found no evidence of cell death, impaired proliferation, or cell cycle arrest, suggesting that ALT activation may prevent oxidative damage from reaching levels that threaten cell survival.


Subject(s)
Chromosomal Instability/drug effects , Fibroblasts/drug effects , Hydrogen Peroxide/pharmacology , Oxidative Stress , Telomere Homeostasis , Telomere/drug effects , Cell Survival , Cellular Senescence/drug effects , Cytokinesis/drug effects , Fetus , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Humans , In Situ Hybridization, Fluorescence , Kinetochores/drug effects , Kinetochores/metabolism , Kinetochores/ultrastructure , Mitosis/drug effects , Primary Cell Culture , Telomere/metabolism , Telomere/ultrastructure , Time-Lapse Imaging
11.
Article in English | MEDLINE | ID: mdl-26520385

ABSTRACT

In recent years, terahertz (THz) radiation has been widely used in a variety of applications: medical, security, telecommunications and military areas. However, few data are available on the biological effects of this type of electromagnetic radiation and the reported results, using different genetic or cellular assays, are quite discordant. This multidisciplinary study focuses on potential genotoxic and cytotoxic effects, evaluated by several end-points, associated with THz radiation. For this purpose, in vitro exposure of human foetal fibroblasts to low frequency THz radiation (0.1-0.15THz) was performed using a Compact Free Electron Laser. We did not observe an induction of DNA damage evaluated by Comet assay, phosphorylation of H2AX histone or telomere length modulation. In addiction, no induction of apoptosis or changes in pro-survival signalling proteins were detected. Moreover, our results indicated an increase in the total number of micronuclei and centromere positive micronuclei induction evaluated by CREST analysis, indicating that THz radiation could induce aneugenic rather than clastogenic effects, probably leading to chromosome loss. Furthermore, an increase of actin polymerization observed by ultrastructural analysis after THz irradiation, supports the hypothesis that an abnormal assembly of spindle proteins could lead to the observed chromosomal malsegregation.


Subject(s)
Actins/metabolism , Centromere/radiation effects , Chromosome Segregation/radiation effects , Fibroblasts/radiation effects , Micronuclei, Chromosome-Defective/statistics & numerical data , Aneuploidy , Apoptosis/radiation effects , Cell Survival/radiation effects , Cells, Cultured , Centromere/genetics , DNA Damage , Fibroblasts/metabolism , Foreskin/cytology , Foreskin/embryology , Histones/metabolism , Humans , In Vitro Techniques , Male , Phosphorylation , Terahertz Radiation
12.
PLoS One ; 9(10): e110963, 2014.
Article in English | MEDLINE | ID: mdl-25354277

ABSTRACT

One main function of telomeres is to maintain chromosome and genome stability. The rate of telomere shortening can be accelerated significantly by chemical and physical environmental agents. Reactive oxygen species are a source of oxidative stress and can produce modified bases (mainly 8-oxoG) and single strand breaks anywhere in the genome. The high incidence of guanine residues in telomeric DNA sequences makes the telomere a preferred target for oxidative damage. Our aim in this work is to evaluate whether chromosome instability induced by oxidative stress is related specifically to telomeric damage. We treated human primary fibroblasts (MRC-5) in vitro with hydrogen peroxide (100 and 200 µM) for 1 hr and collected data at several time points. To evaluate the persistence of oxidative stress-induced DNA damage up to 24 hrs after treatment, we analysed telomeric and genomic oxidative damage by qPCR and a modified comet assay, respectively. The results demonstrate that the genomic damage is completely repaired, while the telomeric oxidative damage persists. The analysis of telomere length reveals a significant telomere shortening 48 hrs after treatment, leading us to hypothesise that residual telomere damage could be responsible for the telomere shortening observed. Considering the influence of telomere length modulation on genomic stability, we quantified abnormal nuclear morphologies (Nucleoplasmic Bridges, Nuclear Buds and Micronuclei) and observed an increase of chromosome instability in the same time frame as telomere shortening. At subsequent times (72 and 96 hrs), we observed a restoration of telomere length and a reduction of chromosome instability, leaving us to conjecture a correlation between telomere shortening/dysfunction and chromosome instability. We can conclude that oxidative base damage leads to abnormal nuclear morphologies and that telomere dysfunction is an important contributor to this effect.


Subject(s)
DNA Damage , Oxidative Stress , Telomere Shortening , Telomere/drug effects , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chromosomal Instability , Humans , Hydrogen Peroxide/toxicity , Telomere/genetics , Telomere/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...