The use of premature chromosome condensation to study in interphase cells the influence of environmental factors on human genetic material

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

Dose assessment intercomparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay)

Purpose: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. Materials and methods: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy c-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. Results: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. Conclusions: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay

RENEB accident simulation exercise

Purpose: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. Materials and methods: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. Results: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). Conclusions: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested

Chromatin dynamics during cell cycle mediate conversion of DNA damage into chromatid breaks and affect formation of chromosomal aberrations: Biological and clinical significance

The formation of diverse chromosomal aberrations following irradiation and the variability in radiosensitivity at different cell-cycle stages remain a long standing controversy, probably because most of the studies have focused on elucidating the enzymatic mechanisms involved using simple DNA substrates. Yet, recognition, processing and repair of DNA damage occur within the nucleoprotein complex of chromatin which is dynamic in nature, capable of rapid unfolding, disassembling, assembling and refolding. The present work reviews experimental work designed to investigate the impact of chromatin dynamics and chromosome conformation changes during cell-cycle in the formation of chromosomal aberrations. Using conventional cytogenetics and premature chromosome condensation to visualize interphase chromatin, the data presented support the hypothesis that chromatin dynamic changes during cell-cycle are important determinants in the conversion of sub-microscopic DNA lesions into chromatid breaks. Consequently, the type and yield of radiation-induced chromosomal aberrations at a given cell-cycle-stage depends on the combined effect of DNA repair processes and chromatin dynamics, which is cell-cycle-regulated and subject to up- or down-regulation following radiation exposure or genetic alterations. This new hypothesis is used to explain the variability in radiosensitivity observed at various cell-cycle-stages, among mutant cells and cells of different origin, or among different individuals, and to revisit unresolved issues and unanswered questions. In addition, it is used to better understand hypersensitivity of AT cells and to provide an improved predictive G2-assay for evaluating radiosensitivity at individual level. Finally, experimental data at single cell level obtained using hybrid cells suggest that the proposed hypothesis applies only to the irradiated component of the hybrid. (C) 2010 Elsevier B.V. All rights reserved

Mitochondria malfunctions as mediators of stem-cells' related carcinogenesis: A hypothesis that supports the highly conserved profile of carcinogenesis

Cancer development is an evolutionary process that has been highly conserved among centuries within organisms. Based on this, the interest in cancer research focuses on cells, organelles and genes that possess a genetic conservatism from yeasts to human. Towards this thought, mitochondria, the highly conserved and responsible for the cellular bioenergetic activity organelles, might play crucial role in carcinogenesis. Interestingly, tumors with low bioenergetic signature have worse prognosis and show a decreased expression of ATPase protein. Furthermore, according to the stem-cell theory of carcinogenesis, aggressive tumors are characterized by an increase number of malignant stem-like cell population and their resistance to chemotherapy has been found to be mitochondrially driven. The above considerations triggered us to hypothesize that mitochondrial bioenergetic processes in stem-like cancer cells plays a crucial role in the highly conserved process of carcinogenesis. Specifically, we support that mitochondrial and/or nuclear DNA alterations that control stem cells’ ATP production drive stem cells to “immortalization” (Otto Warburg theory) that mediates cancer initiation and progression. Substantiation of our hypothesis requires evidence that: (1) alterations in mitochondria bioenergetic metabolites and enzymes encoded either from the mtDNA or the nuclear DNA are linked to human cancer and (2) mitochondrial functions are regulated by highly conserved genes involved in cancer-related cellular processes such as apoptosis, aging and autophagy. Experimental approach on how this hypothesis might be tested and promising strategies in cancer therapeutics are also discussed. In case the hypothesis of stem-cell bioenergetic malformations’ related carcinogenesis proves to be correct, it would contribute to the development of new prognostic, diagnostic and even more effective therapeutic interventions against various types of cancer. (C) 2012 Elsevier Ltd. All rights reserved

The radiosensitizing potential of glutaraldehyde on MCF7 breast cancer cells as quantified by means of the G2-chromosomal radiosensitivity assay

Glutaraldehyde (GA) is a high production volume chemical that is very reactive with a wide spectrum of medical, scientific and industrial applications. Concerning the genotoxic and carcinogenic effect of GA, controversial results have been reported, while in humans no studies with positive carcinogenic results for GA have been published. However, our previous study concerning the combined effects of exposure to both GA and ionising radiation (IR) in peripheral blood lymphocytes of healthy donors has shown that non-genotoxic doses of the chemical induces a statistically significant increase in chromosomal radiosensitivity. The lack of information concerning the radiosensitizing potential of GA on cancerous cells triggered us to test the radiosensitizing effect of GA on breast cancer cells (MCF7). For this purpose the G2-chromosomal radiosensitivity assay (G2-assay) was used. The assay involves G2-phase irradiation and quantitation of the chromosomal fragility in the subsequent metaphase. The experimental data show that 48 h exposure to GA, at doses that are not clastogenic to MCF7 breast cancer cells enhances G2-chromosomal radiosensitivity of this cell line. In an effort to evaluate whether the observed increase in GAs-induced G2-chromosomal radiosensitization is linked to GA-induced alterations in the cell cycle and feedback control mechanism, Mitotic Index analysis was performed. The results have shown that such a mechanism cannot be directly related to the observed GA-induced increase in G2-chromosomal radiosensitivity. Since increased G2-chromosomal radiosensitivity has been linked with cancer proneness, the radiosensitizing effect of GA at non-clastogenic doses highlights its potential carcinogenic profile

The use of premature chromosome condensation to study in interphase cells the influence of environmental factors on human genetic material

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 environments. These factors exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environments 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 G 1 and G 2 phases of the cell cycle. The PCC method has been successfully used in problems involving cell cycle analysis, diagnosis, and prognosis of human leukemia, 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 order to answer questions in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics, and experimental therapeutics. KEYWORDS: premature chromosome condensation, cell fusion, calyculin-A, lymphocytes, DNA damage, genotoxicity, sister chromatid exchanges, chromosomal damage, chromosome aberrations, cell cycle delay, chemicals, ionizing radiation Hatzi et al.: Interphase genotoxicity assessment using PCC TheScientificWorldJOURNAL (2006TheScientificWorldJOURNAL ( ) 6, 1174TheScientificWorldJOURNAL ( -1190 INTRODUCTION Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental agents to which humans are exposed in their natural and anthropogenic environments. Harmful environmental agents, exerting their effect on living organisms from the beginning of life, developed the appropriate evolutionary pressure that resulted in their defense mechanisms. Human technological development as well as human lifestyle introduced new series of harmful agents that, either alone or in combination, affect humans and all other organisms. These new agents exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environments that constitute part of the anthropogenic environment. The most important consequences of human exposure to environmental hazards are DNA damage induced either by direct or indirect binding to DNA. Direct-acting chemicals like DNA alkylating agents (e.g., mitomycin-C), crosslinking agents (e.g., methyl methanesulfonate), and oxygen radicals (e.g., hydrogen peroxide), bind covalently to DNA The exposure of cells to genotoxic factors may result in changes at the chromatid level, such as sister chromatid exchanges (SCEs), as well as in structural chromosomal alterations such as chromosomal aberrations Until now, the conventional cytogenetic analysis of chromosomal damage as a result of exposure to environmental chemicals is mainly based on the microscopic analysis of chromosomes in metaphase In this report, the advantages of the PCC methodologies used as an alternative to conventional metaphase analysis or in combination with other cytogenetic techniques are reviewed. Furthermore, the use of PCC in overcoming problems that could not be solved with the conventional metaphase analysis is Hatzi et al.: Interphase genotoxicity assessment using PCC TheScientificWorldJOURNAL (2006) 6, 1174-1190 1176 presented, and particular emphasis is given to the potential use of PCC to elucidate the mechanisms underlying conversion of DNA damage into chromosomal damage. Additional applications of the PCC methodologies in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics, and experimental therapeutics are described, and future perspectives are discussed. PCC METHODOLOGIES PCC Induction Using Cell Fusion Central for the PCC assay is the fusion of interphase "test" cells with mitotic "inducer" cells that can be mediated either by incubation with Sendai virus As an alternative to the Sendai virus, the well-known fusogen polyethylene glycol (PEG) was applied to PCC-induction procedures Using the PCC assay by means of cell fusion, interphase cells that are either cycling, noncycling, or arrested can be visualized and analyzed. In the hybrids formed by cell fusion, the mitotic factors present in the donor mitotic cell dissolve the nucleus membrane and condense chromatin of the interphase "test" cell. More specifically, cells that have undergone PCC assume a morphology that is characteristic of the position of the interphase cell in the cell cycle: single chromatid per chromosome in G 1 phase, double chromatids per chromosome in G 2 , and pulverized chromosome regions in S phase Chemically Induced PCC Chemically induced PCCs can be obtained by the use of the chemical compounds such as calyculin-A. Calyculin-A was initially isolated from the marine sponge Discodermia calyx 1177 APPLICATIONS OF PCC METHODOLOGIES Biodosimetry and Biomonitoring of Exposure to Ionizing Radiation Exposure of cells to ionizing radiation has been shown to cause a wide variety of phenomena, the most prominent of which are the induction of mutations, the induction of transformation, cell cycle arrest, and cell death. As a result, the study of phenomena elicited by radiation are of particular importance to human health and, therefore, elucidation of the underlying biochemical mechanisms and cytogenetic effects of ionizing radiation are a high priority in radiation biology research. In the field of biological dosimetry, numerous methods capable of detecting radiation-induced changes at the molecular, cytogenetic, and cellular level have been used in order to obtain absorbed dose estimates For these reasons, the PCC methodology was proposed as an alternative biodosimetric tool by Pantelias and Maillie in 1984[50] and since then it has been extensively used to assess and evaluate the induction and repair of chromosome damage after in vivo or in vitro exposure of human cells to ionizing radiation Mechanisms Underlying Conversion of DNA Damage into Chromosomal Damage Visualizing cells with conventional analysis at metaphase can give information concerning only the residual damage after exposure to genotoxic environmental factors. The analysis of such interactions is based, therefore, only on those cells that proceed to mitosis. Therefore, it is difficult to elucidate the mechanism underlying the conversion of DNA damage into chromosomal damage. With the PCC methodologies, it is possible to gain valuable information not only to understand the biochemical mechanisms that affect the conversion of DNA damage into chromosomal damage, but also to determine possibly chromosomal radiosensitivity in G 2 phase, as well as variability in radiosensitivity at various stages of the cell cycle. The Hatzi et al.: Interphase genotoxicity assessment using PCC TheScientificWorldJOURNAL (2006) 6, 1174-1190 1181 onset and the efficiency of chromatin condensation-decondensation are important determinants of these processes. Data obtained so far demonstrate the important role of cdk1/cyclin-B complex and of the G 2 checkpoint control mechanism in affecting chromatin conformation changes and conversion of DNA damage into chromosomal damage. Using the PCC method, it was realized specifically that changes in chromatin conformation soon after irradiation, presumably as a result of histone-H1 phosphorylationdephosphorylation, strongly affected the conversion of DNA lesions into visual PCC fragments. The cdk1/cyclin-B complex was originally defined as the mitosis promoting factor (MPF), identified in mitotic frog eggs as a factor capable of inducing mitosis in G 2 -phase cells. Regulation of cdk1/cyclin-B complexes at multiple levels ensures the tight regulation of the timing of mitotic entry In early reports, G 0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as chromatin condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. From these experiments, it was concluded that changes in chromosome conformation interfered with repair processes of DNA damage. In contrast, resting chromosomes (as G 0 lymphocytes irradiated before fusion) showed efficient repair of chromosomal damage. These results supported the hypothesis that DNA damage is converted into cytogenetic lesions and becomes observable when chromatin conformation changes occur during the cell cycle G 2 checkpoint facilitates repair of chromosomal damage, and the hypothesis that G 2 -checkpoint defects during the G 2 -to M-phase transition can also affect G 2 -chromosomal sensitivity. This was tested using caffeine to abolish G 2 checkpoint by inhibiting ATM protein (Ataxia Telangiectasia mutated protein) Concerning the variability of radiosensitivity to ionizing radiation at various stages of the cell cycle, it is already known that middle to S phase and G 1 phase are resistant stages, while mitosis, G 1 /S, and G 2 /M transition are very sensitive to radiation Genotoxicity of Exposure to Chemical Agents It is well known that increased rates of cell proliferation can escalate the risk of malignancy following exposures to chemical agents 1183 The Uses of PCC in Clinical Cytogenetics and Experimental Therapeutics The fact that chromosomal analysis by means of the PCC method requires a small amount (0.5 ml or less) of sample (i.e., peripheral blood or bone marrow) makes it especially suitable for in vivo and in vitro studies, and also in clinical applications for diagnostic purposes. In particular, the ability to visualize the interphase chromosomes of bone marrow and blood cells using PCC by means of mitotic cell fusion with interphase cells has proved useful and accurate in the study of human acute leukemia Recently, calyculin-A-induced PCC has been combined with multicolor FISH [pq-COBRA-FISH (COmbined Binary RAtio labeling-fluorescence in situ hybridization)] for the cytogenetic analysis of cancer cell lines In the field of prenatal diagnosis, preliminary attempts have been made to combine calyculin-Ainduced PCC with GTG banding for fetus examination Furthermore, a major area of experimental therapeutic research centers on the function of anticancer drugs. In this field, the PCC methodology has been applied to study the effect of several anticancer drugs, such as BCNU, CCNU, Cis-acid, VM-26, adriamycin, and neocarzinostatin, whose mode of action depends on cell cycle arres

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3–1 keV/μm, α-particles 116 keV/μm and 36Ar ions 270 keV/μm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5–16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage