Deliverables Quantitative data on persistence and accumulation of mitochondrial DNA mutations following GI induction A critical role of mitochondrial metabolism is suggested by the lack of signal production by cells, which do not have a functional G6PD enzyme. In vivo pathological consequences of induced instability Overall, the data are consistent with the expression of delayed death and delayed cytogenetic aberrations in the bone marrow being inversely related and reflecting genotypic differences in signalling pathways associated with damage recognition and apoptotic response. A feature of irradiated haemopoietic tissue responses is the generation of long-lived inflammatory-type processes that may produce genotype-dependent bystander-mediated damage. These data are also consistent with the proposition that the genetic background that produces the more effective apoptotic response and phagocytic clearance is less predisposed to adverse consequences of irradiation due to a more effective elimination of unstable and potentially malignant cells. The work with genetic linkage analysis is on going. The current position is that at least three loci are involved in determining whether a cell apoptoses after damage. There is no correlation between radiation-induced delayed genomic instability and radiation-induced cellular or malignant transformation of osteoblastic cells. Changes in gene expression in X-irradiated human lymphocytes at delayed times Concerning bulk cultures, the process of prolonged in vitro culture had a stronger general influence on the global gene expression patterns than the irradiation had. With increasing culture time, a larger number of genes differed in expression between irradiated and control cultures, but there seems not to be a few, readily identifiable genes, which were dramatically affected in all samples. Apart from the indication of a number of individual genes, we have observed a tendency towards a more heterogeneous general gene expression pattern in irradiated cell clones. This may either be a consequence of the genomic instability, where multiple chromosomal lesions lead to changes in the regulation of neighbouring genes or, alternatively, the observed "transcriptomic diversification" could be a manifestation of an epigenetically de-regulated state of the cell with a globally relaxed control of normal regulatory constraints and therefore a more diversified evolution of sub clones in the culture. These properties of the system under study may constitute a model for early events in carcinogenic progression, and the dynamics of gene expression pattern changes in the irradiated clones compared to controls could permit a more comprehensive elucidation of such processes. Furthermore, it was possible to identify a set of marker genes that correlated with the irradiation status of clones and which were also differentially expressed in some of the bulk culture comparisons. This constitutes a set of candidate genes that may be further studied in terms of their possible relevance for the long-term effects on the phenotype of the irradiated cells. Genetic variation in GI response of cultured human bladder mucosa from 300 patients There appears to be individual patient and mouse genotypic variation in the ability of bladder tissue biopsies to produce the bystander factor. Other studies using a range of epithelial cells from normal and tumour cells indicate a wide variation both in the production of the signal and in the expression of the effect. Dose-response relationship at very low doses for acute protective types of cell death such as HRS and apoptosis and late effects such as genomic instability and delayed cell death in cell lines Recent advances in our knowledge of the biological effects of low doses of ionizing radiation have shown two unexpected phenomena (a) a ''bystander effect'' which can be demonstrated at low doses as a transferable factor(s) causing radio-biological effects in unexposed cells, and (b) low-dose Hyper-Radio Sensitivity (HRS) and Increased Radio Resistance (IRR) which can collectively be demonstrated as change in the dose effect relationship, occurring around 0,5-1 Gy of low LET radiation. In both cases, the effect of very low doses is greater than would be predicted by conventional DNA break/repair-based radiobiology. The results show considerable variation in expression of both phenomena and suggest that cell lines with a large bystander effect do not show induced radio resistance. The reverse is also true. This inverse relationship is not clearly related to p53 or tumour status of the cell line. There is an indication that cell lines which have a radiation dose response curve with a wide shoulder, show HRS/IRR and no bystander effect. The results may suggest new approaches to understand factors controlling death or survival sectoring (with consequent possibility of progression to malignancy), at low radiation doses. Repair deficient cell lines may respond to the occurrence of DNA damage in the population by removing large numbers of cells from the proliferating pool. Role of K-shell excitation in instability induction RBEs for cell inactivation and chromosome aberrations are found strongly correlated with the yields of core events in DNA. The role of inner-shell photo ionizations in the high efficiency of heavy ions to induce chromosomal instability is questionable. Experiments on human cells have been designed and similar results were found. In further work, it will be interesting to investigate more deeply the role of core events in the induction of chromosomal aberrations in human cells. The analysis of delayed cell death and chromosomal aberrations induced by inner-shell ionisations seem now to be relevant and might be investigated too. Location of targets for instability and bystander effects within cells and tissue models A significant level of bystander cell killing is observed after nuclear or cytoplasmic irradiation, suggesting that the response observed with nuclear targeting may be due to energy deposited in the underlying cytoplasmic layer. Pilot studies with nuclear targeting show a role for nitric oxide. Targeting cell cytoplasm of human fibroblasts with helium ions leads to the production of micro-nucleated cells, although there is not a direct correlation with ROS production. Studies in tissue systems suggest that proliferation status and premature differentiation may be important responses under bystander conditions. Changes in the lysosomal compartment are seen as early as 5 minutes after radiation with both alpha particles and X-rays, although the pattern of change and presumably the mechanisms involved are not identical. Changes are seen in other subcellular structures and evidence will be sought to show the relationship between the lysosomal and other changes. A significant bystander effects is observed in ureter fragments when these are locally irradiated. The majority of the response of the tissue fragment is in the form of premature differentiation, which may lead to an overall protective effect. The damaged cells produced, may be a consequence of a small increase in the level of spontaneous damage due to a compensatory increase in proliferation. Effects of inhibitors of oxidative stress on GI induction and persistence Increased oxidative stress is a long-term characteristic of progeny of irradiated cells. The increased oxy-radical generation is actually maintained by a signal produced by the irradiated cells into the medium. The data suggest that initiating events in the apoptotic cascade were induced in unexposed cells by a signal produced by irradiated cells. The calcium pulse data suggest that intracellular calcium increase may trigger the pathway. The data suggest that initiating events in the apoptotic cascade are induced in non-irradiated cells by a signal produced by irradiated cells and that this signal can still be produced in the progeny of irradiated cells for several generations. Identification of genes involved in genomic instability In the mouse experiments, the work with genetic linkage analysis is on going. The current position is that at least three loci are involved in determining whether a cell apoptoses after damage. A set of irradiation-related candidate genes have been identified. Downregulation of the repair and cell cycle checkpoint genes and upregulation of an oncogene seem to play a role in events leading to unstable genome. Changes in gene expression after low dose alpha irradiation in human fibroblasts at delayed times Early and delayed gene expression changes were detected in human fibroblast cells after high LET irradiation. Our results on alpha irradiated cells confirmed earlier findings that irradiation causes an immediate upregulation in damage and stress induced genes. Downregulation of the repair and cell cycle checkpoint genes and upregulation of an oncogene seem to play a role in events leading to un-stable genome. The repair can be suggested to be partially switched off allowing unstable genome to be induced. Abrogation of the cell cycle control allows cells to enter into following cell cycle phase even though DNA damage is misrepaired. Activation of an extra cellular matrix degrading oncogene may in turn reduce the control of the neighbouring cells. At delayed time point, in heavy ion irradiated cells, upregulation of mitogenic and cell growth modulating genes were observed. These changes suggest that normal growth control has been compromised. Downregulation of cell cycle controlling and DNA repair genes were detected at delayed time point in heavy ion irradiated cells, suggesting that these changes can be responsible of maintaining chromosomal instability. Persistence of GI in mitochondrial mutant cell lines The first detectable effect in untargeted cells receiving media borne signals from hit cells is a rapid (1-2 min) calcium pulse, which is followed 30min-6hrs later by changes in mitochondrial membrane permeability and induction of reactive oxygen species. Team members have now showed this in both micro beam and medium transfer model systems. Persistent elevation of reactive oxygen species in cells subject to bystander signals would be a plausible epigenetic driver for genomic instability, which fits the facts. Determination of low dose dependency of instability down to the level of a single particle traversal and its LET dependency Long-term, chromosomal instability in human lymphocytes was induced by single-particle irradiation. The results also suggest that a significant contribution to the induction of delayed chromosomal damage in the progeny of human lymphocytes occurs via bystander mechanism. Data sets for variability of methylation changes between clones from surviving irradiated cells Methylation of repetitive DNA and retroviral-associated elements is affected in radiation induced genomic instability. Publications Other (1) Genomic instability and radiation-induced cancer (RADINSTAB) Author(s): SALOMAA S (STUK - Radiation and Nuclear Safety Authority, Helsinki (FI)) Published in: A project summary published in a DG Research brochure: "Understanding the effects of radiation on health". EUR 19959. Luxembourg, EUR-OP, 2002. pp.26-27, 2002, Page(s) 26-27, ISBN 92-894-3840-1 Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
