ANR-05-SEST-0026

The risks to the public and to workers of exposures to very low doses of ionizing radiation is a matter of debate. In this project, we propose to characterize radiosensitivity to such radiation doses of human skin keratinocytes through high throughput technologies. In a first part of the project, we will try to establish new markers of exposure. For that purpose, we will use differentiated keratinocytes. Such keratinocytes are located in the upper layers of human epidermis and they can be removed for bio-dosimetric purposes by tape stripping. We will use dedicated microarrays based on our previous results on low dose keratinocyte response [1]: 2 Gy Radchip and 10 mGy Radchip. These microarrays will be hybridized to RNAs isolated first from cultured differentiated keratinocytes. Then we will prepare RNAs from irradiated hair keratinocytes. In a second part of the project, we will characterize radiosensitivity of the main targets of radiation in epidermis, the basal cells. These cells are composed of two cellular fractions. One of them contains the proliferative keratinocytes termed transiently amplifying cells (TA). We postulate that these cells are radiosensitive and give rise to the early effects of radiation. The second fraction contains a small number of stem cells. We postulate that these stem cells are radioresistant, due to their quiescence and to a high potential of excluding toxic agents or resisting to genotoxic stess. We will prepare the total population of keratinocytes from normal human skin. We will then isolate three fractions through flow cytometry : differentiated, AT and stem keratinocytes. On these cells, we will study : radiosensitivity, antioxidant metabolism, telomeres and telomerase activity, gene profiles after radiation exposure (DNA microarrays), DNA repair activities (DNA repair arrays). Based on our previous results, we also plan to set up the genetic network on GATA3 transcription factor, which might regulate the keratinocyte response to very low radiation doses.