Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

FP5

THE RET PROTO-ONCOGENE Informe resumido

Project ID: QLG1-CT-2001-01646
Financiado con arreglo a: FP5-LIFE QUALITY
País: Italy

RET as a marker of radiation exposure and radiosensivity

Mice of both inbred strains CBA/J and C57/Bl, were analysed, with the COMET-FISH technique specifically designed to detect presence and amount of genomic fragmentation of RET, c-Abl, and p53 genes, following 0, 1, 2 or 4 Gy of X rays exposure. Analyses were performed on PBL withdrawn 30 , 24 hours, and 30 days after exposure, and compared to data coming from COMET determination on the same samples.

Thyroid tissues and whole body examinations of both strand of mice (eight mice for each) were taken from long term 3 Gy irradiated animals.

COMET values were determined by the dedicated freeware software CASP 2.22 (Comet Assay Software Project, University of Wroclaw, Poland). Data have been scored as the mean +/-95% confidential intervals (95% CI), and the 90 percentile algorithm has been applied to the tail moment (TM), percentage of DNA content on tail (%DNA), and tail length (TL). In this study, we stated that only difference between untreated vs. irradiated PBL were discriminated by mean of COMET assay, and no statistical dose response effects can be further recognizable. Moreover, analyses delivered on single mice confirmed both an inter-individual variability and a fluctuation of experimental procedures that hamper the use of COMET for in vivo experiments.

Data coming from experimental points taken from animals sacrificed 24 hours and 30 days after exposure were undistinguishable from controls, thus confirming prompt DNA repair and clone selection of viable cells. On the contrary, COMET-FISH technique specifically directed to detect presence and amount of genomic fragmentation of RET, c-Abl, and p53 genes was able to discriminated in a statistically significant way all doses delivered, when fragmentations were analyzed contextually for RET, cAbl, and TP53 genes. These data were confirmed by Laser confocal microscopy determination. Data coming from experimental points taken at the later points of 24 hours and 30 days after exposure were undistinguishable from controls, probably for viable clone selection, and a prompt DNA repair.

One year following 3 Gy exposure, no evidence of RET cAbl and p53 genes positive fragmentation resulted from thyroid and bone marrow tissues by COMET-FISH. Accordingly, COMET assay alone was absolutely negative to detect any difference between controls and long term irradiated mice. Histochemistry carried out on thyroid samples gave no evidence of tumor progression, both in C57Bl/6 and CBA/J strain of mice, thus hamper further investigation, at least on the basis of this experimental protocol.

Contacto

Roberto AMENDOLA, (Head of Laboratory)
Tel.: +39-063-0486115
Fax: +39-063-0483644
Correo electrónico
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