25 results on '"Fragkos M"'
Search Results
2. Unilateral ovarian absence
- Author
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Tzitzimikas, S., Fragkos, M., Karavida, A., and Mettler, L.
- Published
- 2013
- Full Text
- View/download PDF
3. Are expanded alleles of the FMR1 gene related to unexplained recurrent miscarriages?
- Author
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Fragkos M, Bili H, Ntelios D, GEORGIOS TZIMAGIORGIS, and Bc, Tarlatzis
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Research Article - Abstract
Background: In women with recurrent miscarriages, up to 50 % of those cases remain unexplained. In this study, we evaluated the impact of Cytosine/Guanine/Guanine (CGG) trinucleotide expansions of the fragile-X mental retardation 1 (FMR1) gene in women with unexplained recurrent miscarriages. Methods: This is a prospective case-control pilot study involving 49 women with unexplained recurrent miscarriages and 49 age-matched controls with documented fertility. The case group consisted of women with a history of two or more consecutive miscarriages, in whom no known factor could be identified. The maximum age of recruitment was 40 years. We obtained blood samples that were checked, using polymerase chain reaction with electrophoresis, for the presence of expanded alleles of the FMR1 gene. We further evaluated using sequencing analysis, those women marked as positive. We set the limit at more than 40 repeats. Results: The repeat sizes of CGG expansion in the FMR1 gene differ significantly in the two population groups (p =0.027). We found four women in the miscarriage group and one in the control group positive for carrying premutation alleles (Odds ratio: 4.267, confidence interval: 0.459-39.629). All the positive cases involved intermediate zone carriers. We found no association between the number of abortions each woman had, and her respective CGG repeat number (p =0.255). Conclusions: Many couples are desperately looking for the cause of their recurrent miscarriage suffering. The CGG expanded allele of the FMR1 gene is possibly to be blamed in some of these cases. More studies are needed to support the results of this prototype study. HIPPOKRATIA 2018, 22(3): 132-136.
- Published
- 2019
4. Topographic imaging of an absorbing object in a tissue-like scattering medium using a single source-detector pair
- Author
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Mertiri, M. Raptis, N. Fragkos, M. A. Roditi, E. and Syvridis, D.
- Abstract
Diffuse optical tomography is an emerging biomedical imaging technique, due to its numerous advantages, such as low cost and non-ionizing radiation. In this work, we have developed a very simple setup, which included a single source - photodiode (SP) pair for scanning a sample of water with diluted Intralipid, simulating a biological tissue. LEDs emitting at 470, 525 and 624 nm, as well as a 650 nm Fabry Perot laser, were used as light sources. Scattered light from the sample was detected by the photodiode placed next to the LED. The SP distance could vary and the phantom could be scanned by moving the SP pair in precise, small and automated steps without any intervention during the measurements. Therefore, we obtained measurements from multiple locations on the sample, with just one SP pair. The presented experimental system verified the feasibility of deploying extremely low cost devices for detection and imaging absorbing objects of 1 cm height, placed inside a scattering medium. Maximum depth detection was 2.5 cm. As expected, the quality of the obtained images was degrading, as the object's depth or the scanning step was increasing. Additionally, we developed Monte Carlo simulations of the setup, which achieved good agreement with the experimental results. We also conducted another set of simulations, studying the depth sensitivity of a single static SP pair considering a scattering medium similar to the experimental phantom with and without object. We observed that the depth sensitivity increases as the source wavelength increases from 450 nm to 650 nm.
- Published
- 2019
5. Experimental Evaluation of Modulation Formats' Performance in Diffuse UV Channels
- Author
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Raptis, N. Pikasis, E. Nikas, T. Fragkos, M.-A. Syvridis, D.
- Abstract
The fundamental properties of free space optical channels in the ultraviolet (UV) spectral range between 200 and 280 nm are the enhanced scattering and the solar blind operation. Exploiting these properties, point-to-point links in a non-line-of-sight (NLOS) regime were experimentally tested and the performance of pulse position modulation of fourth order (4-PPM) and flip-orthogonal frequency division multiplexing (Flip-OFDM) modulation formats was evaluated. At the transmitting side, light-emitting diodes (LEDs) emitting at 265 nm were used. At the receiving side, a photo-multiplier tube was combined with an optical filter. Distances up to 20 m were covered and kilobits/s rates were applied. The performance was also evaluated after modifying artificially the atmospheric conditions by the operation of a fog machine. In short, 4-PPM outperformed Flip-OFDM, whereas a medium that offers enriched scattering due to fog may boost the performance of short-range NLOS links in the UV spectral range. © 1989-2012 IEEE.
- Published
- 2017
6. Topographic imaging of an absorbing object in a tissue-like scattering medium using a single source-detector pair.
- Author
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Mertiri, M., Raptis, N., Fragkos, M. A., Roditi, E., and Syvridis, D.
- Published
- 2019
- Full Text
- View/download PDF
7. Topographic imaging of an absorbing object in a tissue-like scattering medium using a single source-detector pair
- Author
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Cullum, Brian M., Kiehl, Douglas, McLamore, Eric S., Mertiri, M., Raptis, N., Fragkos, M. A., Roditi, E., and Syvridis, D.
- Published
- 2019
- Full Text
- View/download PDF
8. Use of the hereditary persistence of fetal hemoglobin 2 enhancer to increase the expression of oncoretrovirus vectors for human gamma-globin
- Author
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Fragkos, M. Anagnou, N.P. Tubb, J. Emery, D.W.
- Subjects
hemic and lymphatic diseases - Abstract
The development of oncoretrovirus vectors for human γ-globin has been hampered by problems of low expression and gene silencing. In order to address these problems, we investigated an enhancer element identified from individuals with deletional hereditary persistence of fetal hemoglobin 2 (HPFH2), a genetic condition characterized by elevated levels of γ-globin in adults. Plasmid transfection studies in erythroid MEL (murine erythroleukemia) cells demonstrated the HPFH2 element could function synergistically with the β-globin locus control region to enhance the expression of an Aγ-globin gene with a truncated -382bp promoter. A series of oncoretrovirus vectors were subsequently generated that contain an expression cassette for Aγ-globin linked to various combinations of the HPFH2 enhancer, the α-globin HS40 enhancer, and several versions of the promoter from Aγ-globin or β-globin. Expression analysis in transduced MEL cell clones revealed very high levels of promoter-autonomous silencing that was at least partially abrogated by the HPFH2 enhancer. The vector containing a combination of a -201 bp Aγ-globin gene promoter with the Greek HPFH -117 point mutation and both the HPFH2 and HS40 enhancers exhibited no signs of vector silencing and was expressed at 248 ± 99% per copy of mouse α-globin (62% of total α-globin). This represents a significant improvement over previously reported oncoretrovirus vectors for Aγ-globin, and demonstrates the capacity of the HPFH2 enhancer to abrogate sequence-autonomous silencing of the Aγ-globin promoter in the context of a gene transfer vector. © 2005 Nature Publishing Group All rights reserved.
- Published
- 2005
9. Unilateral ovarian absence
- Author
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Tzitzimikas, S., primary, Fragkos, M., additional, Karavida, A., additional, and Mettler, L., additional
- Published
- 2012
- Full Text
- View/download PDF
10. The Role of γH2AX in Replication Stress-induced Carcinogenesis: Possible Links and Recent Developments.
- Author
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Fragkos M, Choleza M, and Papadopoulou P
- Abstract
Cancer is a condition characterized by genomic instability and gross chromosomal aberrations. The inability of the cell to timely and efficiently complete its replication cycle before entering mitosis is one of the most common causes of DNA damage and carcinogenesis. Phosphorylation of histone 2AX (H2AX) on S139 (γH2AX) is an indispensable step in the response to DNA damage, as it is required for the assembly of repair factors at the sites of damage. γH2AX is also a marker of DNA replication stress, mainly due to fork collapse that often follows prolonged replication stalling or repair of arrested forks, which involves the generation of DNA breaks. Although the role of γH2AX in the repair of DNA breaks has been well defined, the function of γH2AX in replicative stress remains unclear. In this review, we present the recent advances in the field of replication stress, and highlight a novel function for γH2AX that is independent of its role in the response to DNA damage. We discuss studies that support a role for γΗ2ΑΧ early in the response to replicative stress, which does not involve the repair of DNA breaks. We also highlight recent data proposing that γH2AX acts as a chromatin remodeling component, implicated in the efficient resolution of stalled replication forks. Understanding the mechanism by which γH2AX enables cellular recovery after replication stress will allow identification of novel cancer biomarkers, as well as new targets for cancer therapies., Competing Interests: The Authors have no conflicts of interest to declare in relation to this study., (Copyright 2023, International Institute of Anticancer Research.)
- Published
- 2023
- Full Text
- View/download PDF
11. Dicer prevents genome instability in response to replication stress.
- Author
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Fragkos M, Barra V, Egger T, Bordignon B, Lemacon D, Naim V, and Coquelle A
- Abstract
Dicer, an endoribonuclease best-known for its role in microRNA biogenesis and RNA interference pathway, has been shown to play a role in the DNA damage response and repair of double-stranded DNA breaks (DSBs) in mammalian cells. However, it remains unknown whether Dicer is also important to preserve genome integrity upon replication stress. To address this question, we focused our study on common fragile sites (CFSs), which are susceptible to breakage after replication stress. We show that inhibition of the Dicer pathway leads to an increase in CFS expression upon induction of replication stress and to an accumulation of 53BP1 nuclear bodies, indicating transmission of replication-associated damage. We also show that in absence of a functional Dicer or Drosha, the assembly into nuclear foci of the Fanconi anemia (FA) protein FANCD2 and of the replication and checkpoint factor TopBP1 in response to replication stress is impaired, and the activation of the S-phase checkpoint is defective. Based on these results, we propose that Dicer pre-vents genomic instability after replication stress, by allowing the proper recruitment to stalled forks of proteins that are necessary to maintain replication fork stability and activate the S-phase checkpoint, thus limiting cells from proceeding into mitosis with under-replicated DNA., Competing Interests: CONFLICTS OF INTEREST The authors declare no competing financial interests.
- Published
- 2019
- Full Text
- View/download PDF
12. Are expanded alleles of the FMR1 gene related to unexplained recurrent miscarriages?
- Author
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Fragkos M, Bili H, Ntelios D, Tzimagiorgis G, and Tarlatzis BC
- Abstract
Background: In women with recurrent miscarriages, up to 50 % of those cases remain unexplained. In this study, we evaluated the impact of Cytosine/Guanine/Guanine (CGG) trinucleotide expansions of the fragile-X mental retardation 1 ( FMR1 ) gene in women with unexplained recurrent miscarriages., Methods: This is a prospective case-control pilot study involving 49 women with unexplained recurrent miscarriages and 49 age-matched controls with documented fertility. The case group consisted of women with a history of two or more consecutive miscarriages, in whom no known factor could be identified. The maximum age of recruitment was 40 years. We obtained blood samples that were checked, using polymerase chain reaction with electrophoresis, for the presence of expanded alleles of the FMR1 gene. We further evaluated using sequencing analysis, those women marked as positive. We set the limit at more than 40 repeats., Results: The repeat sizes of CGG expansion in the FMR1 gene differ significantly in the two population groups (p =0.027). We found four women in the miscarriage group and one in the control group positive for carrying premutation alleles (Odds ratio: 4.267, confidence interval: 0.459-39.629). All the positive cases involved intermediate zone carriers. We found no association between the number of abortions each woman had, and her respective CGG repeat number (p =0.255)., Conclusions: Many couples are desperately looking for the cause of their recurrent miscarriage suffering. The CGG expanded allele of the FMR1 gene is possibly to be blamed in some of these cases. More studies are needed to support the results of this prototype study. HIPPOKRATIA 2018, 22(3): 132-136., Competing Interests: The authors declare not having any competing interests., (Copyright 2018, Hippokratio General Hospital of Thessaloniki.)
- Published
- 2018
13. Author Correction: RNAs coordinate nuclear envelope assembly and DNA replication through ELYS recruitment to chromatin.
- Author
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Aze A, Fragkos M, Bocquet S, Cau J, and Méchali M
- Abstract
In the original version of this Article, the affiliation details for Antoine Aze, Michalis Fragkos, Stéphane Bocquet, Julien Cau and Marcel Méchali incorrectly omitted 'CNRS and the University of Montpellier'. This has now been corrected in both the PDF and HTML versions of the Article.
- Published
- 2018
- Full Text
- View/download PDF
14. RNAs coordinate nuclear envelope assembly and DNA replication through ELYS recruitment to chromatin.
- Author
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Aze A, Fragkos M, Bocquet S, Cau J, and Méchali M
- Subjects
- Animals, Cell Cycle drug effects, Cell Cycle genetics, Cell Extracts chemistry, Cell Extracts pharmacology, Cell Nucleus genetics, Cell Nucleus metabolism, Chromatin genetics, Chromatin metabolism, DNA genetics, DNA metabolism, DNA-Binding Proteins genetics, Male, Nuclear Envelope genetics, Nuclear Pore genetics, Nuclear Pore metabolism, Ovum cytology, Ovum metabolism, RNA genetics, Spermatozoa metabolism, Transcription Factors genetics, Xenopus Proteins genetics, Xenopus laevis, DNA Replication, DNA-Binding Proteins metabolism, Nuclear Envelope metabolism, RNA metabolism, Transcription Factors metabolism, Xenopus Proteins metabolism
- Abstract
Upon fertilisation, the sperm pronucleus acquires the competence to replicate the genome through a cascade of events that link chromatin remodelling to nuclear envelope formation. The factors involved have been partially identified and are poorly characterised. Here, using Xenopus laevis egg extracts we show that RNAs are required for proper nuclear envelope assembly following sperm DNA decondensation. Although chromatin remodelling and pre-replication complex formation occur normally, RNA-depleted extracts show a defect in pre-RC activation. The nuclear processes affected by RNA-depletion included ELYS recruitment, which accounts for the deficiency in nuclear pore complex assembly. This results in failure in chromatin relaxation as well as in the import and proper nuclear concentration of the S-phase kinases necessary for DNA replication activation. Our results highlight a translation-independent RNA function necessary for the parental genome progression towards the early embryonic cell cycle programme.
- Published
- 2017
- Full Text
- View/download PDF
15. Rescue from replication stress during mitosis.
- Author
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Fragkos M and Naim V
- Subjects
- Animals, Chromosomal Instability genetics, Chromosome Fragile Sites genetics, Fanconi Anemia genetics, Humans, DNA Replication, Mitosis, Stress, Physiological
- Abstract
Genomic instability is a hallmark of cancer and a common feature of human disorders, characterized by growth defects, neurodegeneration, cancer predisposition, and aging. Recent evidence has shown that DNA replication stress is a major driver of genomic instability and tumorigenesis. Cells can undergo mitosis with under-replicated DNA or unresolved DNA structures, and specific pathways are dedicated to resolving these structures during mitosis, suggesting that mitotic rescue from replication stress (MRRS) is a key process influencing genome stability and cellular homeostasis. Deregulation of MRRS following oncogene activation or loss-of-function of caretaker genes may be the cause of chromosomal aberrations that promote cancer initiation and progression. In this review, we discuss the causes and consequences of replication stress, focusing on its persistence in mitosis as well as the mechanisms and factors involved in its resolution, and the potential impact of incomplete replication or aberrant MRRS on tumorigenesis, aging and disease.
- Published
- 2017
- Full Text
- View/download PDF
16. DNA replication origin activation in space and time.
- Author
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Fragkos M, Ganier O, Coulombe P, and Méchali M
- Subjects
- Animals, Cell Differentiation physiology, Chromosomes, Human genetics, Chromosomes, Human metabolism, DNA genetics, Humans, DNA biosynthesis, DNA Replication physiology, G1 Phase physiology, Replication Origin physiology, S Phase physiology
- Abstract
DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1-S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.
- Published
- 2015
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- View/download PDF
17. Thyrotropin receptor autoantibodies and early miscarriages in patients with Hashimoto thyroiditis: a case-control study.
- Author
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Toulis KA, Goulis DG, Tsolakidou K, Hilidis I, Fragkos M, Polyzos SA, Gerofotis A, Kita M, Bili H, Vavilis D, Daniilidis M, Tarlatzis BC, and Papadimas I
- Subjects
- Abortion, Spontaneous epidemiology, Abortion, Spontaneous etiology, Adult, Case-Control Studies, Female, Gestational Age, Hashimoto Disease complications, Hashimoto Disease epidemiology, Hashimoto Disease immunology, Humans, Middle Aged, Pregnancy, Pregnancy Outcome epidemiology, Risk Factors, Seroepidemiologic Studies, Abortion, Spontaneous blood, Autoantibodies blood, Hashimoto Disease blood, Receptors, Thyrotropin immunology
- Abstract
We have previously hypothesized that early miscarriage in women with Hashimoto thyroiditis might be the result of a cross-reactivity process, in which blocking autoantibodies against thyrotropin receptor (TSHr-Ab) antagonize hCG action on its receptor on the corpus luteum. To test this hypothesis from the clinical perspective, we investigated the presence of TSHr-Ab in Hashimoto thyroiditis patients with apparently unexplained, first-trimester recurrent miscarriages compared to that in Hashimoto thyroiditis patients with documented normal fertility. A total of 86 subjects (43 cases and 43 age-matched controls) were finally included in a case-control study. No difference in the prevalence of TSHr-Ab positivity was detected between cases and controls (Fisher's exact test, p value = 1.00). In patients with recurrent miscarriages, TSHr-Ab concentrations did not predict the number of miscarriages (univariate linear regression, p value = 0.08). These results were robust in sensitivity analyses, including only cases with full investigation or those with three or more miscarriages. We conclude that no role could be advocated for TSHr-Ab in the aetiology of recurrent miscarriages in women with Hashimoto thyroiditis.
- Published
- 2013
- Full Text
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18. Reversible auditory brainstem responses screening failures in high risk neonates.
- Author
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Psarommatis I, Florou V, Fragkos M, Douniadakis E, and Kontrogiannis A
- Subjects
- Hearing Loss, Conductive congenital, Humans, Infant, Infant, Newborn, Risk Factors, Evoked Potentials, Auditory, Brain Stem, Hearing Loss, Conductive diagnosis, Hearing Loss, Sensorineural congenital, Hearing Loss, Sensorineural diagnosis, Neonatal Screening
- Abstract
This work is aimed at assessing the frequency of occurrence of reversible auditory brainstem responses (ABR) abnormalities within a targeted hearing screening program for high risk (HR) newborns. The effect of age on screening is also evaluated and some important clinical issues are highlighted. The audiological records of 1,294 HR neonates were retrospectively reviewed. All children were tested for hearing loss using ABR within a 17-year period. Initial failures were re-examined 4-6 months later. The mean age of infants who scored "pass" and "refer" at initial test, as well as the referral rates were calculated and compared. One hundred and seventy-eight infants (13.8%) demonstrated abnormal recordings at initial screening. From those who were present on re-examination, 64.2% showed complete and 15% partial recovery. Reversible abnormalities have been detected not only for conductive threshold elevation but also for sensorineural losses. Remarkably, 50% of the cases with absent waveforms or ABR threshold ≥ 80 dBnHL demonstrated complete recovery to normal. Statistically, higher rates of abnormal results were inversely associated with the newborn's age at initial testing. In conclusion, reversible ABR abnormalities are common among HR infants due to temporary auditory dysfunction, secondary to external and middle ear pathology or retarded central nervous system maturation. The observed high rates of transient ABR abnormalities give rise to some practical questions regarding the implementation time of hearing screening for HR infants. Moreover, given that central nervous system maturation changes may still be in progress, the definite decision for an early cochlear implantation in this pediatric subset should be made after obtaining reliable behavioral hearing tests.
- Published
- 2011
- Full Text
- View/download PDF
19. Clarithromycin induced reversible sensorineural hearing loss.
- Author
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Hajiioannou JK, Florou V, Kousoulis P, Fragkos M, and Moshovakis E
- Subjects
- Adult, Anti-Bacterial Agents therapeutic use, Clarithromycin therapeutic use, Female, Follow-Up Studies, Hearing drug effects, Hearing Loss, Sensorineural physiopathology, Humans, Respiratory Tract Infections drug therapy, Anti-Bacterial Agents adverse effects, Clarithromycin adverse effects, Hearing Loss, Sensorineural chemically induced
- Abstract
Objective: We present a rare case of reversible sensorineural hearing loss caused by clarithromycin., Methods: We present a case report of hearing loss following clarithromycin administration; we also review the current literature and case reports concerning macrolides and, in particular, clarithromycin induced hearing loss., Results: A young pregnant woman presented with sensorineural hearing loss after clarithromycin intake. The subject's hearing returned to normal limits after drug discontinuation and short-term treatment with low dose steroids., Conclusion: Newer macrolides are considered to be safer regarding ototoxic effects, and a few cases have been previously described. The present case adds to the body of knowledge concerning clarithromycin ototoxicity. Clinicians should be aware of this rare complication and a thorough otologic history should be established prior to macrolide administration. A baseline audiogram and close observation for patients at higher risk is suggested to identify patients with prior hearing loss and serve as baseline for future reference.
- Published
- 2011
20. Mitotic catastrophe occurs in the absence of apoptosis in p53-null cells with a defective G1 checkpoint.
- Author
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Fragkos M and Beard P
- Subjects
- Animals, Autophagy, Caspases metabolism, Cell Line, Tumor, DNA Fragmentation, Dependovirus genetics, Humans, Mice, Models, Biological, Necrosis, Tumor Suppressor Protein p53 metabolism, Apoptosis, G1 Phase Cell Cycle Checkpoints, Mitosis, Tumor Suppressor Protein p53 deficiency
- Abstract
Cell death occurring during mitosis, or mitotic catastrophe, often takes place in conjunction with apoptosis, but the conditions in which mitotic catastrophe may exhibit features of programmed cell death are still unclear. In the work presented here, we studied mitotic cell death by making use of a UV-inactivated parvovirus (adeno-associated virus; AAV) that has been shown to induce a DNA damage response and subsequent death of p53-defective cells in mitosis, without affecting the integrity of the host genome. Osteosarcoma cells (U2OSp53DD) that are deficient in p53 and lack the G1 cell cycle checkpoint respond to AAV infection through a transient G2 arrest. We found that the infected U2OSp53DD cells died through mitotic catastrophe with no signs of chromosome condensation or DNA fragmentation. Moreover, cell death was independent of caspases, apoptosis-inducing factor (AIF), autophagy and necroptosis. These findings were confirmed by time-lapse microscopy of cellular morphology following AAV infection. The assays used readily revealed apoptosis in other cell types when it was indeed occurring. Taken together the results indicate that in the absence of the G1 checkpoint, mitotic catastrophe occurs in these p53-null cells predominantly as a result of mechanical disruption induced by centrosome overduplication, and not as a consequence of a suicide signal.
- Published
- 2011
- Full Text
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21. H2AX is required for cell cycle arrest via the p53/p21 pathway.
- Author
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Fragkos M, Jurvansuu J, and Beard P
- Subjects
- Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cell Line, Cell Nucleus metabolism, Cyclin-Dependent Kinase Inhibitor p21 genetics, DNA Damage, DNA Repair, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Dependovirus genetics, Dependovirus metabolism, Histones genetics, Humans, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, RNA Interference, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins metabolism, Cell Cycle physiology, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Histones metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
Phosphorylation of H2AX (gammaH2AX) is an early sign of DNA damage induced by replication stalling. However, the role of H2AX in the repair of this type of DNA damage is still unclear. In this study, we used an inactivated adeno-associated virus (AAV) to induce a stalled replication fork signal and investigate the function of gammaH2AX. The cellular response to AAV provides a unique model to study gammaH2AX function, because the infection causes pannuclear H2AX phosphorylation without any signs of damage to the host genome. We found that pannuclear gammaH2AX formation is a result of ATR overactivation and diffusion but is independent of ATM. The inhibition of H2AX with RNA interference or the use of H2AX-deficient cells showed that gammaH2AX is dispensable for the formation and maintenance of DNA repair foci induced by stalled replication. However, in the absence of H2AX, the AAV-containing cells showed proteosome-dependent degradation of p21, followed by caspase-dependent mitotic catastrophe. In contrast, H2AX-proficient cells as well as H2AX-complemented H2AX(-/-) cells reacted by increasing p21 levels and arresting the cell cycle. The results establish a new role for H2AX in the p53/p21 pathway and indicate that H2AX is required for p21-induced cell cycle arrest after replication stalling.
- Published
- 2009
- Full Text
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22. Recombinant adeno-associated viral vectors are deficient in provoking a DNA damage response.
- Author
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Fragkos M, Breuleux M, Clément N, and Beard P
- Subjects
- Cell Line, DNA, Viral biosynthesis, Dependovirus radiation effects, Genes, Reporter, Green Fluorescent Proteins biosynthesis, Green Fluorescent Proteins genetics, Humans, Ultraviolet Rays, Virus Replication, DNA Damage, Dependovirus physiology, Genetic Vectors physiology
- Abstract
Adeno-associated virus type 2 (AAV2) provokes a DNA damage response that mimics a stalled replication fork. We have previously shown that this response is dependent on ataxia telangiectasia-mutated and Rad3-related kinase and involves recruitment of DNA repair proteins into foci associated with AAV2 DNA. Here, we investigated whether recombinant AAV2 (rAAV2) vectors are able to produce a similar response. Surprisingly, the results show that both single-stranded and double-stranded green fluorescent protein-expressing rAAV2 vectors are defective in producing such a response. We show that the DNA damage signaling initiated by AAV2 was not due to the virus-encoded Rep or viral capsid proteins. UV-inactivated AAV2 induced a response similar to that of untreated AAV2. This type of DNA damage response was not provoked by other DNA molecules, such as single-stranded bacteriophage M13 or plasmid DNAs. Rather, the results indicate that the ability of AAV2 to produce a DNA damage response can be attributed to the presence of cis-acting AAV2 DNA sequences, which are absent in rAAV2 vectors and could function as origins of replication creating stalled replication complexes. This hypothesis was tested by using a single-stranded rAAV2 vector containing the p5 AAV2 sequence that has previously been shown to enhance AAV2 replication. This vector was indeed able to trigger DNA damage signaling. These findings support the conclusion that efficient formation of AAV2 replication complexes is required for this AAV2-induced DNA damage response and provide an explanation for the poor response in rAAV2-infected cells.
- Published
- 2008
- Full Text
- View/download PDF
23. Chk1 instability is coupled to mitotic cell death of p53-deficient cells in response to virus-induced DNA damage signaling.
- Author
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Jurvansuu J, Fragkos M, Ingemarsdotter C, and Beard P
- Subjects
- Cell Cycle Proteins antagonists & inhibitors, Cell Cycle Proteins metabolism, Cell Death, Cell Line, Checkpoint Kinase 1, Chromosomes genetics, Cyclin B antagonists & inhibitors, Cyclin B metabolism, Cyclin B1, Dependovirus genetics, Enzyme Stability, Metaphase, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins antagonists & inhibitors, Proto-Oncogene Proteins metabolism, Tumor Suppressor Protein p53 genetics, cdc25 Phosphatases antagonists & inhibitors, cdc25 Phosphatases metabolism, Polo-Like Kinase 1, DNA Damage, Dependovirus physiology, Mitosis, Protein Kinases metabolism, Signal Transduction, Tumor Suppressor Protein p53 deficiency
- Abstract
Adeno-associated virus (AAV) DNA, by mimicking a stalled replication fork, provokes a DNA damage response that can arrest cells in the G2/M phase of the cell-cycle. This response depends strictly on DNA damage signaling kinases ATR and Chk1. Here, we used AAV to study long-term effects of DNA damage signaling in cells with altered p53 status. In HCT116 cells, in response to damage signaling, p53 represses transcription of the genes encoding mitotic regulators Cdc25C, cyclin B1, and Plk1 to establish a firm G2 arrest. Isogenic cells lacking p53 maintain these three proteins at constant levels yet can still arrest initially in G2 because Chk1 signaling inhibits their enzymatic activities. Unexpectedly, the levels of Chk1 fall abruptly in a proteasome-dependent manner after two days of arrest in G2. In p53-deficient cells, this Chk1 instability is coupled to recovery of the phosphatase activity of Cdc25C and in the kinase activities of Plk1 and Cdk1/cyclin B1. Consequently, the p53-deficient cells enter lethal mitosis. Thus, the Chk1-mediated arrest is transient: it initially causes cells to accumulate in G2 until p53-dependent transcriptional repression of mitotic proteins takes over. p53-deficient cells cannot maintain the DNA damage signaling-induced G2 arrest after Chk1 has disappeared, and continue into catastrophic mitosis. Restoring Chk1 prevents the cells from entering such mitosis. These results reveal a mechanism based on Chk1 stability that regulates mitotic entry after DNA damage and elucidate the controversial phenomenon of p53-promoted cell survival in the face of damage signaling.
- Published
- 2007
- Full Text
- View/download PDF
24. Hematopoietic stem cell gene therapy.
- Author
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Emery DW, Nishino T, Murata K, Fragkos M, and Stamatoyannopoulos G
- Subjects
- Animals, Gene Transfer Techniques, Genetic Vectors, Humans, Lentivirus genetics, Models, Animal, Spumavirus genetics, Genetic Therapy methods, Hematologic Diseases therapy, Hematopoietic Stem Cell Transplantation methods
- Abstract
Gene therapy applications that target hematopoietic stem cells (HSCs) offer great potential for the treatment of hematologic disease. Despite this promise, clinical success has been limited by poor rates of gene transfer, poor engraftment of modified cells, and poor levels of gene expression. We describe here the basic approach used for HSC gene therapy, briefly review some of the seminal clinical trials in the field, and describe several recent advances directed toward overcoming these limitations.
- Published
- 2002
- Full Text
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25. An araC-controlled bacterial cre expression system to produce DNA minicircle vectors for nuclear and mitochondrial gene therapy.
- Author
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Bigger BW, Tolmachov O, Collombet JM, Fragkos M, Palaszewski I, and Coutelle C
- Subjects
- AraC Transcription Factor, Arabinose genetics, Base Sequence, DNA, Bacterial chemistry, Bacterial Proteins, Cell Nucleus genetics, DNA, Bacterial genetics, Genetic Therapy, Genetic Vectors, Integrases genetics, Mitochondria genetics, Repressor Proteins genetics, Transcription Factors, Viral Proteins
- Abstract
The presence of CpG motifs and their associated sequences in bacterial DNA causes an immunotoxic response following the delivery of these plasmid vectors into mammalian hosts. We describe a biotechnological approach to the elimination of this problem by the creation of a bacterial cre recombinase expression system, tightly controlled by the arabinose regulon. This permits the Cre-mediated and -directed excision of the entire bacterial vector sequences from plasmid constructs to create supercoiled gene expression minicircles for gene therapy. Minicircle yields using standard culture volumes are sufficient for most in vitro and in vivo applications whereas minicircle expression in vitro is significantly increased over standard plasmid transfection. By the simple expedient of removing the bacterial DNA complement, we significantly reduce the size and CpG content of these expression vectors, which should also reduce DNA-induced inflammatory responses in a dose-dependent manner. We further describe the generation of minicircle expression vectors for mammalian mitochondrial gene therapy, for which no other vector systems currently exist. The removal of bacterial vector sequences should permit appropriate transcription and correct transcriptional cleavage from the mitochondrial minicircle constructs in a mitochondrial environment and brings the realization of mitochondrial gene therapy a step closer.
- Published
- 2001
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