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Molecular basis of radiosensitivity

Objective


(i) The single cell microgel (comet) assay has been successfully employed to demonstrate individual sensitivity to induction of radiation induced DNA strand breaks in human T-lymphocytes, and to investigate the role of radical scavengers to counteract strand break formation. The latter has been achieved by modulation of dietary levels of vitamin C in a panel of normal volunteers. In addition, radiation induced cytotoxicity and micronuclei have been assayed to investigate potentially lethal and clastogenic damage to identify radiosensitive individuals in the normal population as well as ataxia-telangiectasia (AT) heterozygotes. With respect to AT, evidence has been gained from a panel of 16 probands, their sibs and parents that mutation frequency in T-cells might be of prognostic value. A collection of radiation sensitive fibroblast cell lines has been established to investigate the possibility that chromosome damage can provide an early measurement of individual radiosensitivity. Three unique radiosensitive individuals have been identified not exhibiting the clinical features of AT. Analysis of their fibroblasts reveals AT-like radiosensitivty and in one case defective double strand break rejoining. These individuals probably carry mutations in human genes (different from AT) which affect radiosensitivity.
In Fanconi anemia (FA) cells an unbalanced cytokine production was demonstrated. In particular abnormalities in tumor necrosis factor alpha TNF(alpha) appear to be a general characteristic associated with FA and FA heterozygous individuals as revealed from a systematic study in FA families. The abnormally high production of TNF(alpha) is also seen in AT cell lines and might contribute to the enhanced apoptotic of FA and AT cell lines. Surprisingly the induction of apoptosis by radiation was drastically reduced in AT and FA compared to normal cells and this was associated with a lack of p53 induction. Altered expression of p53 may affect genes that regulate genomic stability and therefore be relevant to the phenotype of FA and AT syndromes.

(ii) Different and complementary strategies have been used in the different laboratories to study cellular responses to radiation.
the molecular basis of radiation sensitivity in humans. Such knowledge is essential for assessing relative radiation risk between individuals. Increased radiosensitivity may in many cases be found in individuals who are heterozygous for genes controlling defects in DNA repair. The proposal contains three separate projects, which are best presented as one overall programme in order to highlight the comprehensive nature of the Proposal. (1) Identification of radiosensitive individuals will be undertaken both in the normal population and in individuals with known radiosensitive genetic disorders. A battery of complementary techniques will be used to address this problem. The assay systems will be at the molecular, subcellular and cellular levels. One specific problem to be addressed will be the suggestion, for which there is now a great deal of evidence, that about 10% of breast cancer patients are heterozygous for the disorder ataxia-telangiectasia (A-T). A method for identifying A-T heterozygotes on the basis of their mild radiosensitivity is being developed and effort from several contracting laboratories will go into validating the detection systems, and verifying the relationship with breast cancer. (2) Understanding the cellular response to radiation will continue by cloning of human DNA repair genes. It has been now unequivocally demonstrated that basic molecular processes are conserved through the eukaryotic evolutionary ladder, and that the genes involved are conserved between yeast and man. This proven value of the easily manipulated yeast genes as stepping stones to obtaining the homologous human genes will be vigorously pursued. The cloning of genes is a vital step towards identifying their function in repairing radiation damage, and in vitro systems for measuring gene function will be developed. Repair of certain types of DNA damage is not homologously distributed over the genome and such heterogeneity can have major implications for mutation induction. We will continue to investigate the intragenomic heterogeneity of repair of different types of DNA damage to improve understanding of cellular responses. (3) The third project is a study of the tissue and organ-specificity of the response to radiation. This includes the construction of transgenic animals with modified DNA repair genes to study systemic effects of radiation.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Rijksuniversiteit Leiden
Address
72,Wassenaarseweg
2300 RA Leiden
Netherlands

Participants (5)

Erasmus Universiteit Rotterdam
Netherlands
Address
50,Dr. Molewaterplein
3000 DR Rotterdam
GSF-RESEARCH CENTER FOR ENVIRONMENT AND HEALTH
Germany
Address
Ingolstädter Landstrasse 1
85764 Neuherberg
Institut Curie
France
Address
Université De Paris Xi (Université Paris-sud) 15 Rue Georges Clémenceau
91405 Orsay
MRC Cell Mutation Unit
United Kingdom
Address
University Of Sussex Falmer
BN1 9RR Brighton
Rijksuniversiteit Leiden
Netherlands
Address

2333 AA Leiden