Genomic Maintenance & Repair
Monday, October 17, 2005 3:00 PM-5:00 PM Exhibit Hall(PP334) Phosphorylation of histone H2AX in radiation-induced micronuclei.
Medvedeva, Natalia*,1, Panyutin, Igor1, Neumann, Ronald1, 1 Clinical Center, Department of Nuclear Medicine, Bethesda, MD, USA
ABSTRACT- Micronucleus formation is a well-known and widely used tool for assessment of the radiation-induced damage in cells. The mechanism of micronucleus formation is complicated, but it is known that DNA double strand breaks are the precursors for most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early and reliable indicator of DNA double-strand breaks; and it is thought to be a signal for concentration of other repair proteins at the site of damage. In the present work we studied phosphorylation status of the histone H2AX in micronuclei and the correlation between phosphorylated H2AX (
H2AX) foci and localization of other repair factor foci in micronuclei. Chinese hamster ovary (CHO) or primary human fibroblasts (BJ) cells were grown in monolayer, then exposed to different doses of gamma radiation from a Co-60 source. After irradiation cell micronuclei were examined for the presence of H2AX and the correlation of those foci with foci formed in the main nucleus. The preliminary results show that the number of H2AX-positive micronuclei is generally high even in non-irradiated cells and that the ratio of H2AX-positive to H2AX-negative micronuclei is dependent upon the cell line. A fraction of H2AX-positive micronuclei demonstrates a dose-dependent increase and reaches a maximum at about 5 hours after irradiation. The analysis of co-localization of H2AX and other repair protein foci shows that despite the presence of 53BP1 at the site of H2AX in the main nucleus, there is no 53BP1 signal in H2AX-positive micronuclei. The results allow us to draw a preliminary conclusion that active DNA repair does not occur in micronuclei, although the high percentage of phosphorylated H2AX indicates persistent changes in the micronucleus chromatin.
Key words: micronuclei, h2ax, dna repair