The Experts below are selected from a list of 1134 Experts worldwide ranked by ideXlab platform

William F Blakely - One of the best experts on this subject based on the ideXlab platform.

  • a novel parameter cell cycle progression index for radiation dose absorbed estimation in the Premature Chromosome Condensation assay
    Radiation Protection Dosimetry, 2014
    Co-Authors: Tomisato Miura, Akifumi Nakata, Kosuke Kasai, Manabu Nakano, Yu Abe, Eiki Tsushima, Natalia I Ossetrova, Mitsuaki A Yoshida, William F Blakely
    Abstract:

    Abstract The calyculin A-induced Premature Chromosome Condensation (PCC) assay is a simple and useful method for assessing the cell-cycle distribution in cells, since calyculin A induces Chromosome Condensation in various phases of the cell cycle. In this study, a novel parameter, the cell-cycle progression index (CPI), in the PCC assay was validated as a novel biomarker for biodosimetry. Peripheral blood was drawn from healthy donors after informed consent was obtained. CPI was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60)Co-gamma rays: ∼0.6 Gy min(-1), or X ray: 1.0 Gy min(-1); 0-10 Gy) model. The calyculin A-induced PCC assay was performed for Chromosome preparation. PCC cells were divided into the following five categories according to cell-cycle stage: non-PCC, G1-PCC, S-PCC, G2/M-PCC and M/A-PCC cells. CPI was calculated as the ratio of G2/M-PCC cells to G1-PCC cells. The PCC-stage distribution varied markedly with irradiation doses. The G1-PCC cell fraction was significantly reduced, and the G2/M-PCC cell fraction increased, in 10-Gy-irradiated PBL after 48 h of culture. CPI levels were fitted to an exponential dose-response curve with gamma-ray irradiation [y = 0.6729 + 0.3934 exp(0.5685D), r = 1.0000, p < 0.0001] and X-ray irradiation [y = -0.3743 + 0.9744 exp(0.3321D), r = 0.9999, p < 0.0001]. There were no significant individual (p = 0.853) or gender effects (p = 0.951) on the CPI in the human peripheral blood ex vivo irradiation model. Furthermore, CPI measurements are rapid (< 15 min per case). These results suggest that the CPI is a useful screening tool for the assessment of radiation doses received ranging from 0 to 10 Gy in radiation exposure early after a radiation event, especially after a mass-casualty radiological incident.

  • optimization of calyculin a induced Premature Chromosome Condensation assay for Chromosome aberration studies
    Cytometry Part A, 2011
    Co-Authors: Tomisato Miura, William F Blakely
    Abstract:

    Calyculin A-induced Premature Chromosome Condensation (PCC) assay is a simple and useful method to assess structural and numerical Chromosome aberrations in cells. Our hypothesis in this study is that suboptimum calyculin A induction of PCC resulting in fuzzy compactness and/or shortened length Chromosomes would decrease the detection sensitivity of numerical and structural Chromosome aberrations such as small PCC rings and small excess fragments. In this study, an optimization of calyculin A exposure on Chromosome morphology and PCC induction frequency was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation (60Co-γ rays; ∼0.6 Gy/min; 0–30 Gy) model. Treatment with calyculin A (50 nM) for 15 and 30 min resulted in 11.3 ± 2.7 and 9.9 ± 1.6-fold increases in the frequency of G2/M-PCC cells with extended length Chromosomes compared with the 60-min treated group over a broad dose range (0 to 20 Gy), respectively. The G2/M-PCC scoring index per PCC in 15- and 30-min treated groups was increased by 1.9 ± 0.2 (P = 0.001) and 1.8 ± 0.2 (P = 0.001) compared with the 60-min treated group over 0–20 Gy, respectively. The G2/M-PCC efficiency of 30-min treated group was highest in the three conditions (i.e., 15-, 30-, and 60-min treatment) of calyculin A exposure. Calyculin A (50 nM) treatment for 30 min before the 48-h harvest of mitogen-stimulated human PBL is optimum for the formation of suitable Chromosome morphology necessary to assess structural Chromosome aberrations induced by exposure to radiation using the chemical induced-PCC assay. Published 2011 Wiley Periodicals, Inc.

  • Premature Chromosome Condensation in human resting peripheral blood lymphocytes for Chromosome aberration analysis using specific whole Chromosome dna hybridization probes
    Methods of Molecular Biology, 2005
    Co-Authors: Pataje G S Prasanna, William F Blakely
    Abstract:

    : This paper describes a unique, simple, and rapid method for inducing Premature Chromosome Condensation (PCC) in "resting" human peripheral blood lymphocytes (HPBLs) and also explains an approach to studying numerical changes and/or structural aberrations involving specific Chromosomes. HPBLs are isolated from whole blood on a density gradient and, to induce PCC, are incubated at 37 degrees C in cell culture medium supplemented with a phosphatase inhibitor (okadaic acid or calyculin A), adenosine triphosphate (ATP), and p34cdc2/cyclin B kinase (an essential component of mitosis-promoting factor [MPF]). PCC spreads are prepared on glass slides after a brief hypotonic treatment of cells and fixing in acetic acid/methanol fixative. Aberrations involving specific Chromosomes are analyzed after in situ hybridization and Chromosome painting by fluorescence microscopy. Normal (undamaged) cells display two fluorescent spots per Chromosome, whereas aneuploid cells, or cells with a structural aberration involving the specific Chromosome corresponding to the painting probe, may show more than two spots. This method may be used in many biological and toxicological fields that require analysis of numerical and structural aberrations involving specific Chromosomes.

  • Premature Chromosome Condensation assay for biodosimetry studies with fission neutrons
    Health Physics, 1997
    Co-Authors: Pataje G S Prasanna, Christopher J Kolanko, H M Gerstenberg, William F Blakely
    Abstract:

    Characterization of the Premature Chromosome Condensation assay for radiation quality is needed. To that end, human lymphocytes were exposed in vitro to various doses of 250-kVp x rays (Y(D) = 4 keV microm(-1), Y(D) is the dose-mean lineal energy of the absorbed dose distribution, D(y), where y is defined as the energy deposited in a volume by a single event divided by the mean chord length of the volume) and to fission neutrons (Y(D) = 65 keV microm(-1)). The distribution of Prematurely condensed Chromosome and fragments following exposure to x rays or to neutrons were non-Poisson after repair at 37 degrees C for 24 h. Dose-response curves were constructed for the yield of excess Prematurely condensed Chromosome fragments as necessary for biodosimetry applications. The curves were fitted to a weighted linear model by the least-squares regression method. The neutron relative biological effectiveness (RBE) value was estimated to be 2.4 +/- 0.39.

  • application of the Premature Chromosome Condensation assay in simulated partial body radiation exposures evaluation of the use of an automated metaphase finder
    Stem Cells, 1995
    Co-Authors: William F Blakely, Pataje G S Prasanna, Christopher J Kolanko, Mark D Pyle, D M Mosbrook
    Abstract:

    The Premature Chromosome Condensation (PCC) assay has been proposed as a useful and rapid end point for biological dosimetry following accidental high-dose radiation overexposures. A major benefit of the PCC assay is that it does not require cells to divide for evaluation of cytogenetic damage. The PCC assay was performed on isolated human peripheral lymphocytes exposed in vitro to doses from 1 to 9 Gy of 250 kVp x-rays. The dose-response relationships of the frequency distribution and the yield of PCC fragments in cells were determined after one day of repair at 37 degrees C. A Qpcc approach, which involves the analysis of the yield of excess PCC fragments in damaged cells, was used to establish a dose-response calibration curve. This method is identical in concept to the Qdr technique introduced by Sasaki for partial-body exposure dose-estimates using asymmetrical Chromosome aberrations (i.e., dicentrics and rings) in metaphase spreads of human lymphocytes. A simulated in vitro test of a partial-body exposure to a 6-Gy dose was performed. The results from this test provided dose estimates of 5.3 +/- 0.6, 4.7 +/- 0.6, 5.0 +/- 0.6 and 4.7 +/- 0.8 Gy for the 20, 30, 50 and 75 percent component of 6-Gy irradiated cells, respectively. An automated metaphase-finding system was evaluated for use with the PCC assay. This system helped to locate PCC spreads among the mitotic inducer Chinese hamster ovary (CHO) metaphase spreads, thereby facilitating rapid scoring of samples.(ABSTRACT TRUNCATED AT 250 WORDS)

George Iliakis - One of the best experts on this subject based on the ideXlab platform.

  • investigation of bystander effects in hybrid cells by means of cell fusion and Premature Chromosome Condensation induction
    Radiation Research, 2010
    Co-Authors: Georgia I Terzoudi, George Iliakis, C Dontabakoyianni, Gabriel E Pantelias
    Abstract:

    The established dogma in radiation sciences that underlies radiation protection and therapeutic applications is that radiation effects require induction of DNA damage only in cells that are directly hit by the radiation. However, extensive work during the last decade demonstrates that DNA damage responses can be detected in cells that are only bystanders. Such effects include cell killing and responses associated with DNA and Chromosome damage. Here, we developed a strategy for investigating bystander effects on chromosomal integrity by Premature Chromosome Condensation using hybrid cell formation between nontargeted human lymphocytes and targeted CHO cells or vice versa. We reasoned that signaling molecules generated in the targeted component of the hybrid will transfer to the nontargeted cell, inducing damage detectable at the chromosomal level. The results indicate that bystander cytogenetic effects between CHO and human lymphocytes cannot be detected under the experimental conditions used. This may be due either to the lack of communication of such responses between the components of the hybrid or to their abrogation by the experimental manipulations. These observations and the methodology developed should be useful in the further development of protocols for investigating bystander responses and for elucidating the underlying mechanisms.

  • Premature Chromosome Condensation reveals dna pk independent pathways of Chromosome break repair
    Radioprotection, 2008
    Co-Authors: Georgia I Terzoudi, Gabriel E Pantelias, Satyendra K Singh, George Iliakis
    Abstract:

    Cells of higher eukaryotes process double strand breaks (DSBs) in their genome using a non-homologous end joining apparatus that utilizes DNA-PK and other well characterized factors (D-NHEJ). Cells with defects in D-NHEJ, repair the majority of DSBs using a slow-repair pathway which is independent of genes of the RAD52 epistasis group and functions as a backup (B-NHEJ). Recent studies implicate DNA ligase III, PARP-1 and histone H1 in this pathway of NHEJ. The present study investigates the operation of B-NHEJ in the repair of interphase Chromosome breaks visualized in irradiated G0 human lymphocytes by Premature Chromosome Condensation (PCC). Chromosome breaks are effectively repaired in human lymphocytes, but repair is significantly compromised after treatment with wortmannin, a DNA-PK inhibitor. Despite slower kinetics, cells exposed to wortmannin rejoin the majority of ionizing radiation-induced Chromosome breaks suggesting that B-NHEJ is also functional at the Chromosome level. Complementation of D-NHEJ defect in wortmannin-treated lymphocytes by newly made DNA-PK is only possible under conditions of nuclear envelop break down and Premature Chromosome Condensation, suggesting that in interphase cells the shunting of Chromosome breaks from D-NHEJ to B-NHEJ is irreversible.

  • mitosis promoting factor activity of inducer mitotic cells may affect radiation yield of interphase Chromosome breaks in the Premature Chromosome Condensation assay
    Cancer Research, 1993
    Co-Authors: Xinbo Cheng, Gabriel E Pantelias, Ryuichi Okayasu, Nge Cheong, George Iliakis
    Abstract:

    We measured mitosis-promoting factor (MPF) activity in two cell lines, CHO and HeLa, extensively used at mitosis as inducers in the assay of Premature Chromosome Condensation to study the yield and the repair kinetics of radiation damage in interphase Chromosomes of diverse cell lines. We found a 2.5-fold higher MPF activity in HeLa as compared to CHO mitotic cells per mg of crude extract protein. HeLa mitotic cells, when used as inducers of Premature Chromosome Condensation, uncovered two times more interphase Chromosome breaks in irradiated, nonstimulated human lymphocytes as compared to CHO mitotic cells. A 2-fold increase in the yield of interphase Chromosome breaks with HeLa mitotics was also observed in G1 cells from plateau-phase CHO cultures. Thus, MPF activity may be a contributing factor of the process that transforms radiation-induced DNA damage to Chromosome breaks, and subsequently to other types of lethal Chromosome aberrations. We speculate that the level and the control in the cell cycle of MPF activity may influence the radiosensitivity of cells to killing. The results strongly suggest that a direct comparison between the yields of interphase Chromosome breaks measured in different laboratories may not be possible unless similar inducer cells with similar MPF activity are used.

  • hypertonic treatment during Premature Chromosome Condensation allows visualization of interphase Chromosome breaks repaired with fast kinetics in irradiated cho cells
    Radiation Research, 1993
    Co-Authors: George Iliakis, Ryuichi Okayasu, John M Varlotto, Claire Shernoff, Ya Wang
    Abstract:

    A class of interphase Chromosome breaks was visualized in irradiated (10 Gy) plateau-phase CHO cells after treatment (2-30 min) in hypertonic (500 mM NaCl) growth medium during the period normally allowed for Chromosome Condensation, in the Premature Chromosome Condensation (PCC) assay. Rejoining of this class of interphase Chromosome breaks was fast $(t_{1/2}=1.5\ {\rm min})$ compared to the rejoining of interphase Chromosome breaks normally observed in the absence of hypertonic treatment $(t_{1/2}=76\ {\rm min})$, suggesting that they are formed from a different subset of precursor DNA lesions. A fast $(t_{1/2\ {\rm fast}}=12\ {\rm min})$ and a slow $(t_{1/2\ {\rm slow}}=71\ {\rm min})$ component were also observed in the rejoining of radiation-induced (50 Gy) DNA double-strand breaks (DSBs), as measured by pulsed-field gel electrophoresis. We propose that fast-repairing DSBs are the precursor lesions underlying the fast-repairing interphase Chromosome breaks observed in these experiments. Slowly repair...

  • Premature Chromosome Condensation reveals dna pk independent pathways of Chromosome break repair
    International Journal of Oncology, 1992
    Co-Authors: Georgia I Terzoudi, Gabriel E Pantelias, Satyendra K Singh, George Iliakis
    Abstract:

    Cells of higher eukaryotes process double strand breaks (DSBs) in their genome using a non-homologous end joining apparatus that utilizes DNA-PK and other well characterized factors (D-NHEJ). Cells with defects in D-NHEJ, repair the majority of DSBs using a slow-repair pathway which is independent of genes of the RAD52 epistasis group and functions as a backup (B-NHEJ). Recent studies implicate DNA ligase III, PARP-1 and histone H1 in this pathway of NHEJ. The present study investigates the operation of B-NHEJ in the repair of interphase Chromosome breaks visualized in irradiated G0 human lymphocytes by Premature Chromosome Condensation (PCC). Chromosome breaks are effectively repaired in human lymphocytes, but repair is significantly compromised after treatment with wortmannin, a DNA-PK inhibitor. Despite slower kinetics, cells exposed to wortmannin rejoin the majority of IR induced Chromosome breaks suggesting that B-NHEJ is also functional at the Chromosome level. Complementation of D-NHEJ defect in wortmannin-treated lymphocytes by newly made DNA-PK is only possible under conditions of nuclear envelope break down and Premature Chromosome Condensation, suggesting that in interphase cells the shunting of Chromosome breaks from D-NHEJ to B-NHEJ is irreversible. The understanding of chromosomal aberration formation allows mechanistic explanations for the carcinogenic potential of D-NHEJ defects.

D M Mosbrook - One of the best experts on this subject based on the ideXlab platform.

  • application of the Premature Chromosome Condensation assay in simulated partial body radiation exposures evaluation of the use of an automated metaphase finder
    Stem Cells, 1995
    Co-Authors: William F Blakely, Pataje G S Prasanna, Christopher J Kolanko, Mark D Pyle, D M Mosbrook
    Abstract:

    The Premature Chromosome Condensation (PCC) assay has been proposed as a useful and rapid end point for biological dosimetry following accidental high-dose radiation overexposures. A major benefit of the PCC assay is that it does not require cells to divide for evaluation of cytogenetic damage. The PCC assay was performed on isolated human peripheral lymphocytes exposed in vitro to doses from 1 to 9 Gy of 250 kVp x-rays. The dose-response relationships of the frequency distribution and the yield of PCC fragments in cells were determined after one day of repair at 37 degrees C. A Qpcc approach, which involves the analysis of the yield of excess PCC fragments in damaged cells, was used to establish a dose-response calibration curve. This method is identical in concept to the Qdr technique introduced by Sasaki for partial-body exposure dose-estimates using asymmetrical Chromosome aberrations (i.e., dicentrics and rings) in metaphase spreads of human lymphocytes. A simulated in vitro test of a partial-body exposure to a 6-Gy dose was performed. The results from this test provided dose estimates of 5.3 +/- 0.6, 4.7 +/- 0.6, 5.0 +/- 0.6 and 4.7 +/- 0.8 Gy for the 20, 30, 50 and 75 percent component of 6-Gy irradiated cells, respectively. An automated metaphase-finding system was evaluated for use with the PCC assay. This system helped to locate PCC spreads among the mitotic inducer Chinese hamster ovary (CHO) metaphase spreads, thereby facilitating rapid scoring of samples.(ABSTRACT TRUNCATED AT 250 WORDS)

  • application of the Premature Chromosome Condensation assay in simulated partial body radiation exposures evaluation of the use of an automated metaphase finder
    Stem Cells, 1995
    Co-Authors: William F Blakely, Pataje G S Prasanna, Christopher J Kolanko, Mark D Pyle, D M Mosbrook
    Abstract:

    Abstract : The Premature Chromosome Condensation (PCC) assay has been proposed as a useful and rapid end point for biological dosimetry following accidental high-dose radiation overexposures. A major benefit of the PCC assay is that it does not require cells to divide for evaluation of cytogenetic damage. The PCC assay was performed on isolated human peripheral lymphocytes exposed in vitro to doses from 1 to 9 Gy of 250 kVp x-rays. The dose-response relationships of the frequency distribution and the yield of PCC fragments in cells were determined after one day of repair at 37 deg C. A Q(pcc) approach, which involves the analysis of the yield of excess PCC fragments in damaged cells, was used to establish a dose-response calibration curve. This method is identical in concept to the Q(dr) technique introduced by Sasaki for partial-body exposure dose-estimates using asymmetrical Chromosome aberrations (i.e., dicentrics and rings) in metaphase spreads of human lymphocytes. A simulated in vitro test of a partial-body exposure to a 6-Gy dose was performed. The results from this test provided dose estimates of 5.3 + or = 0.6, 4.7 + or = 0.6,5.0 + or = 0.6 and 4.7 + or = 0.8 Gy for the 20,30,50 and 75 percent component of 6-Gy irradiated cells, respectively. An automated metaphase-finding system was evaluated for use with the PCC assay. This system helped to locate PCC spreads among the mitotic inducer Chinese hamster ovary (CHO) metaphase spreads, thereby facilitating rapid scoring of samples. We conclude that the measurement of excess PCC fragments in Giemsa-stained preparations provides useful biological dosimetry information on the size of the irradiated fraction in cases of acute radiation exposures. Use of Q(pcc) analysis is recommended for partial-body exposures to determine dose estimates for the irradiated fraction. Automated metaphasc finding significantly enhances the spced of the PCC assay.

Georgia I Terzoudi - One of the best experts on this subject based on the ideXlab platform.

  • Interphase Cytogenetic Analysis of Micronucleated and Multinucleated Cells Supports the Premature Chromosome Condensation Hypothesis as the Mechanistic Origin of Chromothripsis
    Cancers, 2019
    Co-Authors: Antonio Pantelias, Ioanna Karachristou, Alexandros G. Georgakilas, Georgia I Terzoudi
    Abstract:

    The discovery of chromothripsis in cancer genomes challenges the long-standing concept of carcinogenesis as the result of progressive genetic events. Despite recent advances in describing chromothripsis, its mechanistic origin remains elusive. The prevailing conception is that it arises from a massive accumulation of fragmented DNA inside micronuclei (MN), whose defective nuclear envelope ruptures or leads to aberrant DNA replication, before main nuclei enter mitosis. An alternative hypothesis is that the Premature Chromosome Condensation (PCC) dynamics in asynchronous micronucleated cells underlie Chromosome shattering in a single catastrophic event, a hallmark of chromothripsis. Specifically, when main nuclei enter mitosis, Premature chromatin Condensation provokes the shattering of Chromosomes entrapped inside MN, if they are still undergoing DNA replication. To test this hypothesis, the agent RO-3306, a selective ATP-competitive inhibitor of CDK1 that promotes cell cycle arrest at the G2/M boundary, was used in this study to control the degree of cell cycle asynchrony between main nuclei and MN. By delaying the entrance of main nuclei into mitosis, additional time was allowed for the completion of DNA replication and duplication of Chromosomes inside MN. We performed interphase cytogenetic analysis using asynchronous micronucleated cells generated by exposure of human lymphocytes to γ-rays, and heterophasic multinucleated Chinese hamster ovary (CHO) cells generated by cell fusion procedures. Our results demonstrate that the PCC dynamics during asynchronous mitosis in micronucleated or multinucleated cells are an important determinant of Chromosome shattering and may underlie the mechanistic origin of chromothripsis.

  • rapid assessment of high dose radiation exposures through scoring of cell fusion induced Premature Chromosome Condensation and ring Chromosomes
    Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2013
    Co-Authors: A Lamadrid I Boada, Georgia I Terzoudi, Romero I Aguilera, J Gonzalez E Mesa, Gabriel E Pantelias, O E Garcia
    Abstract:

    Abstract Analysis of Premature Chromosome Condensation (PCC) mediated by fusion of G0-lymphocytes with mitotic CHO cells in combination with rapid visualization and quantification of rings (PCC-Rf) is proposed as an alternative technique for dose assessment of radiation-exposed individuals. Isolated lymphocytes or whole blood from six individuals were γ-irradiated with 5, 10, 15 and 20 Gy at a dose rate of 0.5 Gy/min. Following either 8- or 24-h post-exposure incubation of irradiated samples at 37 °C, Chromosome spreads were prepared by standard PCC cytogenetic procedures. The protocol for PCC fusion proved to be effective at doses as high as 20 Gy, enabling the analysis of ring Chromosomes and excess PCC fragments. The ring frequencies remained constant during the 8–24-h repair time; the pooled dose relationship between ring frequency (Y) and dose (D) was linear: Y = (0.088 ± 0.005) × D. During the repair time, excess fragments decreased from 0.91 to 0.59 chromatid pieces per Gy, revealing the importance of information about the exact time of exposure for dose assessment on the basis of fragments. Compared with other cytogenetic assays to estimate radiation dose, the PCC-Rf method has the following benefits: a 48-h culture time is not required, allowing a much faster assessment of dose in comparison with conventional scoring of dicentrics and rings in assays for chemically-induced Premature Chromosome Condensation (PCC-Rch), and it allows the analysis of heavily irradiated lymphocytes that are delayed or never reach mitosis, thus avoiding the problem of saturation at high doses. In conclusion, the use of the PCC fusion assay in conjunction with scoring of rings in G0-lymphocytes offers a suitable alternative for fast dose estimation following accidental exposure to high radiation doses.

  • investigation of bystander effects in hybrid cells by means of cell fusion and Premature Chromosome Condensation induction
    Radiation Research, 2010
    Co-Authors: Georgia I Terzoudi, George Iliakis, C Dontabakoyianni, Gabriel E Pantelias
    Abstract:

    The established dogma in radiation sciences that underlies radiation protection and therapeutic applications is that radiation effects require induction of DNA damage only in cells that are directly hit by the radiation. However, extensive work during the last decade demonstrates that DNA damage responses can be detected in cells that are only bystanders. Such effects include cell killing and responses associated with DNA and Chromosome damage. Here, we developed a strategy for investigating bystander effects on chromosomal integrity by Premature Chromosome Condensation using hybrid cell formation between nontargeted human lymphocytes and targeted CHO cells or vice versa. We reasoned that signaling molecules generated in the targeted component of the hybrid will transfer to the nontargeted cell, inducing damage detectable at the chromosomal level. The results indicate that bystander cytogenetic effects between CHO and human lymphocytes cannot be detected under the experimental conditions used. This may be due either to the lack of communication of such responses between the components of the hybrid or to their abrogation by the experimental manipulations. These observations and the methodology developed should be useful in the further development of protocols for investigating bystander responses and for elucidating the underlying mechanisms.

  • Premature Chromosome Condensation reveals dna pk independent pathways of Chromosome break repair
    Radioprotection, 2008
    Co-Authors: Georgia I Terzoudi, Gabriel E Pantelias, Satyendra K Singh, George Iliakis
    Abstract:

    Cells of higher eukaryotes process double strand breaks (DSBs) in their genome using a non-homologous end joining apparatus that utilizes DNA-PK and other well characterized factors (D-NHEJ). Cells with defects in D-NHEJ, repair the majority of DSBs using a slow-repair pathway which is independent of genes of the RAD52 epistasis group and functions as a backup (B-NHEJ). Recent studies implicate DNA ligase III, PARP-1 and histone H1 in this pathway of NHEJ. The present study investigates the operation of B-NHEJ in the repair of interphase Chromosome breaks visualized in irradiated G0 human lymphocytes by Premature Chromosome Condensation (PCC). Chromosome breaks are effectively repaired in human lymphocytes, but repair is significantly compromised after treatment with wortmannin, a DNA-PK inhibitor. Despite slower kinetics, cells exposed to wortmannin rejoin the majority of ionizing radiation-induced Chromosome breaks suggesting that B-NHEJ is also functional at the Chromosome level. Complementation of D-NHEJ defect in wortmannin-treated lymphocytes by newly made DNA-PK is only possible under conditions of nuclear envelop break down and Premature Chromosome Condensation, suggesting that in interphase cells the shunting of Chromosome breaks from D-NHEJ to B-NHEJ is irreversible.

  • the use of Premature Chromosome Condensation to study in interphase cells the influence of environmental factors on human genetic material
    The Scientific World Journal, 2006
    Co-Authors: Vasiliki I Hatzi, Georgia I Terzoudi, Christina Paraskevopoulou, Vasilios Makropoulos, Demetrios P Matthopoulos, Gabriel E Pantelias
    Abstract:

    Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental factors to which humans are exposed in their natural and anthropogenic environment. These factors exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environment that constitute part of the anthropogenic environment. It is well known that genetic damage due to these factors has dramatic implications for human health. Since most of the environmental genotoxic factors induce arrest or delay in cell cycle progression, the conventional analysis of Chromosomes at metaphase may underestimate their genotoxic potential. Premature Chromosome Condensation (PCC) induced either by means of cell fusion or specific chemicals, enables the microscopic visualization of interphase Chromosomes whose morphology depends on the cell cycle stage, as well as the analysis of structural and numerical aberrations at the G1 and G2 phases of the cell cycle. The PCC has been successfully used in problems involving cell cycle analysis, diagnosis and prognosis of human leukaemia, assessment of interphase Chromosome malformations resulting from exposure to radiation or chemicals, as well as elucidation of the mechanisms underlying the conversion of DNA damage into chromosomal damage. In this report, particular emphasis is given to the advantages of the PCC methodology used as an alternative to conventional metaphase analysis in answering questions in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics and experimental therapeutics.

Gabriel E Pantelias - One of the best experts on this subject based on the ideXlab platform.

  • rapid assessment of high dose radiation exposures through scoring of cell fusion induced Premature Chromosome Condensation and ring Chromosomes
    Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2013
    Co-Authors: A Lamadrid I Boada, Georgia I Terzoudi, Romero I Aguilera, J Gonzalez E Mesa, Gabriel E Pantelias, O E Garcia
    Abstract:

    Abstract Analysis of Premature Chromosome Condensation (PCC) mediated by fusion of G0-lymphocytes with mitotic CHO cells in combination with rapid visualization and quantification of rings (PCC-Rf) is proposed as an alternative technique for dose assessment of radiation-exposed individuals. Isolated lymphocytes or whole blood from six individuals were γ-irradiated with 5, 10, 15 and 20 Gy at a dose rate of 0.5 Gy/min. Following either 8- or 24-h post-exposure incubation of irradiated samples at 37 °C, Chromosome spreads were prepared by standard PCC cytogenetic procedures. The protocol for PCC fusion proved to be effective at doses as high as 20 Gy, enabling the analysis of ring Chromosomes and excess PCC fragments. The ring frequencies remained constant during the 8–24-h repair time; the pooled dose relationship between ring frequency (Y) and dose (D) was linear: Y = (0.088 ± 0.005) × D. During the repair time, excess fragments decreased from 0.91 to 0.59 chromatid pieces per Gy, revealing the importance of information about the exact time of exposure for dose assessment on the basis of fragments. Compared with other cytogenetic assays to estimate radiation dose, the PCC-Rf method has the following benefits: a 48-h culture time is not required, allowing a much faster assessment of dose in comparison with conventional scoring of dicentrics and rings in assays for chemically-induced Premature Chromosome Condensation (PCC-Rch), and it allows the analysis of heavily irradiated lymphocytes that are delayed or never reach mitosis, thus avoiding the problem of saturation at high doses. In conclusion, the use of the PCC fusion assay in conjunction with scoring of rings in G0-lymphocytes offers a suitable alternative for fast dose estimation following accidental exposure to high radiation doses.

  • investigation of bystander effects in hybrid cells by means of cell fusion and Premature Chromosome Condensation induction
    Radiation Research, 2010
    Co-Authors: Georgia I Terzoudi, George Iliakis, C Dontabakoyianni, Gabriel E Pantelias
    Abstract:

    The established dogma in radiation sciences that underlies radiation protection and therapeutic applications is that radiation effects require induction of DNA damage only in cells that are directly hit by the radiation. However, extensive work during the last decade demonstrates that DNA damage responses can be detected in cells that are only bystanders. Such effects include cell killing and responses associated with DNA and Chromosome damage. Here, we developed a strategy for investigating bystander effects on chromosomal integrity by Premature Chromosome Condensation using hybrid cell formation between nontargeted human lymphocytes and targeted CHO cells or vice versa. We reasoned that signaling molecules generated in the targeted component of the hybrid will transfer to the nontargeted cell, inducing damage detectable at the chromosomal level. The results indicate that bystander cytogenetic effects between CHO and human lymphocytes cannot be detected under the experimental conditions used. This may be due either to the lack of communication of such responses between the components of the hybrid or to their abrogation by the experimental manipulations. These observations and the methodology developed should be useful in the further development of protocols for investigating bystander responses and for elucidating the underlying mechanisms.

  • Premature Chromosome Condensation reveals dna pk independent pathways of Chromosome break repair
    Radioprotection, 2008
    Co-Authors: Georgia I Terzoudi, Gabriel E Pantelias, Satyendra K Singh, George Iliakis
    Abstract:

    Cells of higher eukaryotes process double strand breaks (DSBs) in their genome using a non-homologous end joining apparatus that utilizes DNA-PK and other well characterized factors (D-NHEJ). Cells with defects in D-NHEJ, repair the majority of DSBs using a slow-repair pathway which is independent of genes of the RAD52 epistasis group and functions as a backup (B-NHEJ). Recent studies implicate DNA ligase III, PARP-1 and histone H1 in this pathway of NHEJ. The present study investigates the operation of B-NHEJ in the repair of interphase Chromosome breaks visualized in irradiated G0 human lymphocytes by Premature Chromosome Condensation (PCC). Chromosome breaks are effectively repaired in human lymphocytes, but repair is significantly compromised after treatment with wortmannin, a DNA-PK inhibitor. Despite slower kinetics, cells exposed to wortmannin rejoin the majority of ionizing radiation-induced Chromosome breaks suggesting that B-NHEJ is also functional at the Chromosome level. Complementation of D-NHEJ defect in wortmannin-treated lymphocytes by newly made DNA-PK is only possible under conditions of nuclear envelop break down and Premature Chromosome Condensation, suggesting that in interphase cells the shunting of Chromosome breaks from D-NHEJ to B-NHEJ is irreversible.

  • the use of Premature Chromosome Condensation to study in interphase cells the influence of environmental factors on human genetic material
    The Scientific World Journal, 2006
    Co-Authors: Vasiliki I Hatzi, Georgia I Terzoudi, Christina Paraskevopoulou, Vasilios Makropoulos, Demetrios P Matthopoulos, Gabriel E Pantelias
    Abstract:

    Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental factors to which humans are exposed in their natural and anthropogenic environment. These factors exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environment that constitute part of the anthropogenic environment. It is well known that genetic damage due to these factors has dramatic implications for human health. Since most of the environmental genotoxic factors induce arrest or delay in cell cycle progression, the conventional analysis of Chromosomes at metaphase may underestimate their genotoxic potential. Premature Chromosome Condensation (PCC) induced either by means of cell fusion or specific chemicals, enables the microscopic visualization of interphase Chromosomes whose morphology depends on the cell cycle stage, as well as the analysis of structural and numerical aberrations at the G1 and G2 phases of the cell cycle. The PCC has been successfully used in problems involving cell cycle analysis, diagnosis and prognosis of human leukaemia, assessment of interphase Chromosome malformations resulting from exposure to radiation or chemicals, as well as elucidation of the mechanisms underlying the conversion of DNA damage into chromosomal damage. In this report, particular emphasis is given to the advantages of the PCC methodology used as an alternative to conventional metaphase analysis in answering questions in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics and experimental therapeutics.

  • mitosis promoting factor activity of inducer mitotic cells may affect radiation yield of interphase Chromosome breaks in the Premature Chromosome Condensation assay
    Cancer Research, 1993
    Co-Authors: Xinbo Cheng, Gabriel E Pantelias, Ryuichi Okayasu, Nge Cheong, George Iliakis
    Abstract:

    We measured mitosis-promoting factor (MPF) activity in two cell lines, CHO and HeLa, extensively used at mitosis as inducers in the assay of Premature Chromosome Condensation to study the yield and the repair kinetics of radiation damage in interphase Chromosomes of diverse cell lines. We found a 2.5-fold higher MPF activity in HeLa as compared to CHO mitotic cells per mg of crude extract protein. HeLa mitotic cells, when used as inducers of Premature Chromosome Condensation, uncovered two times more interphase Chromosome breaks in irradiated, nonstimulated human lymphocytes as compared to CHO mitotic cells. A 2-fold increase in the yield of interphase Chromosome breaks with HeLa mitotics was also observed in G1 cells from plateau-phase CHO cultures. Thus, MPF activity may be a contributing factor of the process that transforms radiation-induced DNA damage to Chromosome breaks, and subsequently to other types of lethal Chromosome aberrations. We speculate that the level and the control in the cell cycle of MPF activity may influence the radiosensitivity of cells to killing. The results strongly suggest that a direct comparison between the yields of interphase Chromosome breaks measured in different laboratories may not be possible unless similar inducer cells with similar MPF activity are used.