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(PP314) Interstitial -H2AX foci in metaphase chromosomes after irradiation of wild-type and repair deficient G1 cells.
Kato, Takamitsu*,1, Nagasawa, Hatsumi1, Bedoford, Joel, 1 Department of Environmental & Radiological Health Sciences, Ft. Collins, Colorado, USA
ABSTRACT- -H2AX foci that develop in cells shortly after irradiation closely reflect the number of DNA dsbs. These foci also disappear with time after irradiation. We have shown that the effect of some of the damage leading to -H2AX foci that developed in cells irradiated in G1, persists and was observed as foci present interstitially in metaphase chromosomes after cells progressed through S, G2, and into mitosis. Wild-type CHO cells, and a repair deficient XR-1 CHO mutant were studied after irradiation of cells synchronized in G1 by isoleucine deprivation. Cells were either subcultured immediately or after a 12-hour. After subculture, cells were harvested in their first post-irradiation mitosis and the -H2AX focus assay was carried out. The majority of foci were interstitially located in the metaphase chromosomes, and not at the ends. Some appeared as focus-pairs with one member on each chromatid at the same locus, while others appeared on one chromatid only. After 1 Gy, a mean of 10 total foci and 0.7 focus-pairs per cell was seen for immediate subculture, while there were 6 total and 0.3 focus-pairs per cell for delayed subculture. For XR-1 cells after 1 Gy delivered in G1 there were approximately 50 total foci and about 9.7 focus-pairs per cell for both immediate and delayed subculture. This result was similar to parallel studies where the development and disappearance of -H2AX foci were studied in these cells during G1. Shortly after 1 Gy irradiation (20 minutes) the number of foci per cell was 20 for CHO and 25 for XR1. The number decreased to about 2 per cell for CHO cells by 12 hours after irradiation but for XR1 cells the number was about 12 per cell. It is not clear why the number of foci and focus-pairs do not decrease when measured in mitotic XR-1 cells while the number decreases by about half in these cells during G1. However, clearly the chromatin distortions following a dsb in G1, or whatever it is that causes the massive phosphorylation of H2AX, can in some cases be replicated and appears at the same locus on both chromatids during mitosis. Further, there are in some cases more foci in mitotic cells following G1 irradiation than were present in the G1. This suggests the presence of some other DNA damage that is not initially expressed as a dsb but results in the development of a dsb later through S phase into mitosis.
Key words: -H2AX, Chromosome
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