Objective
Aim of the proposed research project is to understand the molecular mechanisms by which radiation-induced changes in the feedback control of the cell cycle affect the evolution from the initial radiation-induced DNA damage into permanent cellular modifications expressed as reproductive cell death, chromosomal aberrations, mutations, malignant transformation, and changes in developmental and differentiation pattern.
The project is designed to test the hypothesis that radiation-induced perturbations of the cell cycle feedback or checkpoint control mechanisms are major determinants in the evolution of radiation damage and therefore of human radiation sensitivity. We propose to study the molecular changes in cyclin/cyclin-dependent protein kinase (cdk) complexes, which regulate cell cycle progression, in response to ionizing radiation.
These complexes, will be analysed for molecular modifications and turnover of their components (cyclins, cdks) and their enzymatic activity. Further goal of the proposed experiments is, to uncover modifying factors regulating cyclin/cdk activity after radiation by affinity purification techniques and functional assays and therefore to search, in the case of the radiation-induced G2-block, for a functional mammalian homologue to the yeast rad9 gene rather than for a sequence homologue.
To study the importance of cell-type specific factors, like chromatin structure and cell-type specific feedback mechanisms, cell cycle-associated changes in chromatin conformation after radiation will be analysed in cells with different radiation sensitivity using the premature chromosome condensation (PCC) technique. The cell cycle associated changes in chromatin and the process of PCC are predominantly a result of the activity of cyclin/cdk complexes. Our working hypotheses is that the higher the cyclin/cdk activity is in a cell during PCC, the more interphase chromosome breaks in irradiated cells will be uncovered. Cyclin/cdk activity in cell fusion hybrids will be determined and be correlated with the extent of initial radiation-induced DNA damage transformed into chromosomal damage as uncovered by PCC. To study the role of different levels of cyclin/cdk activity in the cellular response to radiation, we will, by molecular biology means, develop a cell system in which expression of cyclin/cdk subunits is controllable.
The experiments will be performed on primary human keratinocytes, human peripheral lymphocytes, permanent human cell lines, and on one- and two-cell mouse embryos.
Fundamental to the project is the comparison between primary human cells of ectodermal origin with permanent human cell lines. This comparison will yield information in how far such immortal cells with altered feedback mechanisms are valid general models.
The level of radiation-induced initial DNA damage in somatic as well as embryonic cells will be visualised with the comet assay. By using early mouse embryos, we will be able to study the role of chromatin conformation in actively transcribed genome regions compared to silent regions as well as their repair kinetics. The embryonic genome in mice is not switched on before the second embryonic cell cycle.
The realisation of the proposed project depends on the collaboration of the five participating laboratories. By combining the special expertise of each of the laboratories in cell biology, embryology, biochemistry, cytogenetics, and molecular biology, we will be able to analyse the evolution of radiation-induced genetic damage in relation to feedback control mechanisms of the cell cycle and the cellular consequences from different angles, not possible by a single laboratory.
The proposed studies will shed new light on the evolution of genetic damage induced by ionizing radiation and its relevance to low dose irradiation and, probably, on the nature of the variability of human radiation sensitivity. A full understanding of the processes that lead to cellular damage after radiation and the elucidation of the molecular mechanisms involved as well as the identification of the main factors that define the response to irradiation on the cellular level are of basic importance for radiation protection.
Programme(s)
Call for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
85764 OBERSCHLEISSHEIM
Germany