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Contenuto archiviato il 2024-04-19

Cell cycle checkpoints and the biological effectiveness of DNA damage; molecular dosimetry through the cell cycle

Obiettivo

The objectives of the project are to determine the toxic and mutagenic effects of different types of DNA damage, caused by various classes of genotoxic agents, in different phases of the cell cycle. The type of DNA damage which will be investigated are (i) alkylation products introduced by methylating and ethylating agents, (ii) cyclobutane pyrimidine dimers and (6-4) photoproducts introduced by ultraviolet light (254 nm) and (iii) the DNA adducts introduced by 2-acetoxy acetyl amino fluorene, which causes primarily adducts at the C8-position of guanine.


Cells in organs of experimental animals and in man are often in a resting phase (G0 phase) of the cell cycle. However, in some organs, e.g. bone marrow or intestinal epithelium, cells are cycling rapidly, which resembles more closely the situation of cells cultured in vivo. The toxic and mutagenic effects of different classes of genotoxic agents on cells in different parts of the cell cycle will be investigated. Furthermore, the mechanisms underlying the differential response of cells will be analyzed using cells either deficient in certain steps of DNA repair processes or mutated in genes which are involved in the regulation of the cell cycle. The types of cells to be used in this project are (i) normal human fibroblasts in culture and repair deficient cell lines obtained from patients suffering from repair deficiency syndromes, (ii) Chinese hamster cells, including a set of repair deficient cell lines belonging to different complementation groups and (iii) cells from the yeast Schizosaccharomyces pombe from which repair deficient mutants are available as well as cell strains mutated in cell cycle checkpoints.

In order to study cell cycle effects of genotoxic agents, it is essential to generate cell populations in which all cells are in the same phase of the cell cycle (synchronized populations). To this end human diploid fibroblasts will be grown to confluency and kept at this stage. All cells in such a population will be in the G0 phase. Similarly, Chinese hamster cells will be synchronized by phase. S. pombe cells will be synchronised by centrifugation procedures using an elutriator rotor. G2 phase cells are recovered using this procedure, and their passage through the cell cycle will be monitored by microscopy. The effects of exposure to genotoxic agents will be studied during discrete parts of the cell cycle. Fission yeast cells also enter a G0-like state when nutrients are limiting. This will enable the cellular response to genotoxic agents to be studied in cells that are not dividing. Once the wild type response to DNA damaging agents has been established, the effects of removing individual checkpoints and DNA response pathways by use of appropriate mutants will be studied. It is anticipated that this will help to understand the protective role of specific checkpoints after DNA damage. Comparison of the yeast and mammalian results will help to understand the consequences of exposure to DNA damaging agents in all eukaryotes.

Campo scientifico (EuroSciVoc)

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Coordinatore

Rijksuniversiteit Leiden
Contributo UE
Nessun dato
Indirizzo
72,Wassenaarseweg
2300 RA Leiden
Paesi Bassi

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