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Mechanisms of formation of ionising radiation-induced chromosomal aberrations: Impact of repair pathways and nuclear architecture

Objective

Chromosomal aberrations are efficiently induced by ionising radiation and contribute to a great extent to the development of cancer. Increased resolution of molecular cytogenetics along with the availability of cell lines and knockout mouse models sensitive to radiation, provide the basis for further unravelling of the mechanisms of formation of chromosomal aberrations. The proposal aims at studying the initial DNA damage, its repair and the biological factor influencing the ultimate yield of chromosomal aberrations. To reach this goal different strategies will be undertaken including exploitation of novel technologies, site-specific induction of DNA strand breaks and the use of cells with defined defects in repair. The outcome of this study will contribute to understanding of the mechanisms of chromosomal aberration formation and will ultimately help to extrapolate to the effects low doses and low dose-rates.
In this project we have made main achievements with regard to the understanding of the formation of ionizing radiation induced CA particularly emphasising the role of nuclear architecture in the formation of CA. The most important findings are summarized:
-The repair of DSB displays a fast and slow component. The fraction of radiation induced DSB that is very fast repaired, might represent repair of damage possible related to free radicals induced DNA lesions in open chromatin. The nature of the slow repairing DSB fraction is unclear but unlikely to be related to complex damage (clustered damage) induced by free radicals.
-Immunostaining of histone H2aX-P provides a very sensitive tool to measure DSB at single cell level in a variety of tissues.
-Gene density/transcriptional activity in mammalian chromosomes has no major influence on the formation of CA.
-The genome wide distribution of transcription coupled repair resembles the transcriptome.
-Repair of DNA damage generated by 125I decay (locally multiple damage sites) leads to CA involving unexposed chromosomes supporting the concept that CA can be generated by interaction between damaged and non-damaged DNA (proposed by Chadwick and Leenhouts (1978)).
-Msh2 mismatch repair protein and Werner syndrome helicase play a critical role in the repair of IR induced DSB by homologous recombination in the S-phase (stalled replication) and G2 phase respectively.
-In this project we obtained accumulating evidence that radiosensitivity affects the length of the telomeres, and vice versa, both in vitro studies as well as in vivo (breast cancer patients).
-Ionizing radiation does not induce global changes in nuclear organisation of chromatin.
-IR induces local changes in chromatin involving a genetically controlled redistribution of heterochromatin (chromosomes 1 and 9).
-The positioning of gene rich and gene poor chromosomes predicts the outcome in CA in X-irradiated lymphocytes hence providing for the first time evidence for an impact of nuclear architecture on CA formation.
-We obtained clear evidence that apoptosis affects the outcome of CA after IR. Moreover apoptotic responses appear to be associated with cells carrying unstable aberrations (such as dicentrics).
-Mitogen stimulation of lymphocytes (often used in cytogenetical studies investigating radiation effects) may inhibit apoptosis thereby affecting the frequencies and types of CA after ionising radiation.
-Evidence for a role of p53 in G2 checkpoint after radiation; mutation or suppression of p53 will increase the progression of damaged cells through G2 and affects the outcome of CA.
-Neither chromosome length, gene density nor chromatin conformation affect the persistence of CA in vivo and in vitro. Hence, these factors are not of importance for retrospective biodosimetry.
-Using various molecular probes, the quantification and qualification of CA has reached a high level of resolution allowing to detect efficiently a variety of inter- and intra- changes.
-Interstitial fragments are a hallmark of exposure to high LET radiation. The research that has been granted by this proposal has resulted in a total of 68 peer reviewed publications

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

LEIDEN UNIVERSITY
Address
Wassenaarseweg 72
2300 RA Leiden
Netherlands

Participants (6)

ACADEMISCH ZIEKENHUIS BIJ DE UNIVERSITEIT VAN AMSTERDAM
Netherlands
Address
Meibergdreef 15
1105 AZ Amsterdam
AUTONOMOUS UNIVERSITY OF BARCELONA
Spain
Address
Campus De Bellaterra
08193 Bellaterra
BRUNEL UNIVERSITY
United Kingdom
Address
Kingston Lane
Uxbridge
RUPRECHT-KARLS-UNIVERSITAET HEIDELBERG
Germany
Address
Albert-überle-strasse 3-5
69120 Heidelberg
STOCKHOLM UNIVERSITET
Sweden
Address
Stockholm University
106 91 Stockholm
UNIVERSITÀ DEGLI STUDI DELLA TUSCIA
Italy
Address
Via S. Camillo De Lellis
01100 Viterbo