Deliverables Interstitial telomeric sequence stability; DNA damage, DNA repair and gene amplification Main scientific achievements: There are three types of ITS in the human genome: Short ITS are also present in the hamster genome. Short ITS likely originated during the repair of DNA double strand breaks (DSBs) in the germ line. Short human ITS are not radiosensitive. Repair of DSBs is preferentially associated with deletions at the break site; insertions are rare events and mainly involve few nucleotides. Hamster cell lines containing amplified DNA are frequently radiosensitive, they show spontaneous and radio-induced chromosomal instability. Large ITS are involved more frequently than expected in spontaneous but not in radio-induced chromosomal aberrations. Defects in DSB repair genes increase the probability of occurrence of gene amplification. Gamma-rays and hydrogen peroxide induce gene amplification; the induction is greater in DSB repair deficient cells, confirming a role for DSBs in the initiation of gene amplification. In human tumours different mechanisms can contribute to gene amplification. DNA-PKcs deficiency increases the frequency of telomeric fusions in immortalised MEFs. Technical achievements: Human and hamster cell lines suitable to efficiently study DNA modifications introduced during DSB repair. Analysis of radiation sensitivity using DNA Breakage Detection-Fluorescence In Situ Hybridisation (DBD-FISH) procedure Main scientific achievements: -5 bp human classical satellite DNA sequences contain a high concentration of alkali-labile sites, and also show a higher sensitivity to X-ray induced DNA breakage. -Extensive telomere shortening, as in the fourth generation of knock out mice for telomerase, results in a slower repair rate of ionising radiation-induced double-strand breaks. -Interstitial Telomeric Sequence (ITS) blocks from Chinese hamster cells show a particular chromatin structure that results in a high density of alkali-labile sites, which may explain their sensitivity to breakage and recombination at the chromosomal level. - ITS from Chinese hamster cells exhibit a slower rejoining rate of radiation-induced DNA double-strand breaks compared to that of the whole genome, that is not influenced by DNA-PKcs activity. Technical achievements: -Technical development of the DBD-FISH procedure to detect and quantify DNA damage, adapted for human, mouse, and Chinese hamster cells. -Simultaneous quantification of DNA single- and double-strand breaks, in single cells, in the whole genome or within specific DNA sequence areas. Telomere instability and the formation and transmission of radiation induced DNA damage The aim of TELORAD is to study telomere instability and the formation and transmission of radiation induced DNA damage. TELORAD shows that telomere radio sensitivity and telomere capping are significant steps on the formation and the transmission of radiation-induced damage. Clear associations with DNA damage response and tumour development are emerging in respect of chromosomal instability associated with the structure/ function of the telomeric repeat sequences that cap the ends of chromosomes. Telomere metabolism is closely linked with certain DNA repair proteins and shortening and alteration of telomeres can trigger apoptotic response and/or chromosomal instability and/or amplification via post-irradiation cycles of telomeric fusion-breakage-fusion. The heterogeneity of telomere maintenance intra and inter individual let suspect an important role in cancer susceptibility. It remains to evaluate the impact of such findings on low-level radiation risk estimation and individual radiation-induced risk. This would contribute to the advance of knowledge in radioprotection, an essential prerequisite for the safe use of ionising radiations. Mouse models to study the role of telomeres in cancer, ageing and DNA repair Mammalian cells (mice with genetic backgrounds lacking telomerase, PARP, ATM, DNA-PKs, Ku 80, double mutant mice, human or hamster cells over-expressing specific genes) are the tools used to address the questions of interconnections between the non-homologous end-joining pathway and telomere maintenance. The main scientific achievements are: - Establishment of a murine model that shows chromosomal instability due to critical telomere shortening: Terc KO mice. - Short telomeres result in increased sensitivity to ionising radiation. This is not due to a defect in the major DNA repair pathways (HR and NHEJ), but is related to having a short telomere per se. - The NHEJ proteins Ku and DNA-PKcs have a crucial role in telomere capping, as well as in regulating telomerase action at the telomere. - NHEJ is a crucial pathway in detecting, processing and signalling telomere dysfunction. - DNA-PKcs play a role in maintaining telomeres in cooperation with telomerase activity. Technical breakthroughs: Use of combined SKY/Q-FISH technology for identification of the involvement of telomeres in chromosomal aberrations. Capping of radiation-induced DNA breaks in different telomerase and DNA repair cell environments Main scientific achievements: - Shortened telomeres in telomerase knockout mice join to radiation-induced DNA breaks, thus making correct break repair difficult, and explaining the reported increase in radiation sensitivity of telomerase knockout mice with short telomeres. - The chromosome aberrations resulting from short telomeres joined to DNA breaks may contribute to generating chromosome instability during tumorigenesis in human cells. - An important fraction of DNA breaks in A-T cells do not join other broken chromosome ends. - No evidence for the de novo synthesis of telomeric sequences at broken chromosome ends has been obtained either in A-T cells or from the telomerase knockout study. - Dysfunctional long telomeres in DNA-PKcs knockout mouse join to DNA breaks, but this type of interference of dysfunctional telomeres in DNA break repair contributes only marginally to the increased radiation sensitivity of the DNA-PKcs knockout mouse model. - In spite of the obvious NHEJ deficiency, cells without DNA-PKcs are able to join the broken DNA ends of the chromosomes. Technical achievement: We have established a new assay by which to analyse the induction and persistence of aneuploidies in binucleated cells at interphase. This assay can be applied to aneuploidies resulting from telomere shortening or dysfunction in mice. Telomeres and radiosensitivity Main scientific achievements: - Telomere maintenance as an indicator of radiosensitivity: Human and mouse cells with short telomeres appear to be more sensitive to radiation than their counterparts with longer telomeres. - Interstitial telomeric sites in murine scid cells are not sensitive to radiation: Several interstitial telomeric sites monitored and none of them was sensitivie to radiation. Restoration of DNA-PKcs which is deficient in scid cells resulted in chromosome breakage at interstitial telomeric sites. - Chinese hamster cells deficient in Ku and DNA-PKcs do not show alterations in telomere maintenance. - Preliminary results that the BRCA2 gene may be involved in telomere maintenance. Technical achievements: - Implementation of two techniques for telomere length measurement: Q-FISH and flow-FISH. Telomere maintenance and chromosome instability Mouse radiosensitivity is correlated with telomere length. Human telomeric position effect is determined by chromosomal context and telomeric chromatin integrity. - Inhibition of telomerase by TRF1 and activation of telomere degradation by TRF2. - Human ITS are not radiosensitive. - The interstitial (TTAGGG) repeats do not underlie spontaneous or radiation-induced chromosome aberrations, but rather the structure, the condensation/decondensation or the modification of the chromatin conformation could be involved in chromosome sensitivity. - DNA amplification by breakage/fusion/bridge cycles is initiated by spontaneous telomere loss in a human cancer cell line. Technical achievement: Easy and quick technique to measure telomere length. Publications Other (1) Telomere instability and the formation and transmission of radiation induced DNA damage (TELORAD) Author(s): SABATIER L (Commissariat à l'Energie Atomique, Fontenay-aux-Roses (FR)) 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.30-31, 2002, Page(s) 30-31, ISBN 92-894-3840-1 Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
