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Purpose: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G(0)-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 gamma-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 24h 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 G(0)-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.

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.

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 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.

Checkpoint abrogation in G₂ compromises repair of DNA double-strand breaks (DSB) and confers enhanced G₂ chromosomal radiosensitivity in ataxia telangiectasia (AT) cells. To directly test this hypothesis, we combined calyculin A-induced premature chromosome condensation with conventional cytogenetics to evaluate chromosome damage before and after the G₂ checkpoint in irradiated primary AT and normal human lymphocytes and their lymphoblastoid derivatives. Direct analysis of radiation damage in G₂ by premature chromosome condensation reveals practically indistinguishable levels of chromosomal breaks in AT and normal cells. Yet a 4-fold increase in metaphase chromosome damage is observed in AT cells as compared with normal cells which, in contrast to AT cells, exhibit a strong G₂ arrest manifest as an abrupt reduction in the mitotic index. Thus, an active checkpoint facilitates repair of chromosomal breaks in normal cells. Treatment with caffeine that abrogates the G₂ checkpoint without significantly affecting DSB rejoining increases metaphase chromosome damage of normal cells to the AT level but leaves unchanged interphase chromosome damage in G₂. Caffeine has no effect on any of these end points in AT cells. These observations represent the first direct evidence that the G₂ checkpoint facilitates repair of chromosome damage, presumably by supporting repair of DNA DSBs. Failure to arrest will lead to chromatin condensation and conversion of unrepaired DNA DSBs to chromosomal breaks during G ₂-to-M phase transition. ©2005 American Association for Cancer Research.