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(PP083) DNA damage clusters in bystander cells.
Sutherland, Betsy*,1, Bennett, Paula 1, Cuomo, Noelle1, Yang, Hongying2, Held, Kathryn2, 1 Biology Department, Upton, NY, US2 Department of Radiation Oncology, Boston, MA, US
ABSTRACT- Bystander cells sharing medium with irradiated cells show altered growth, chromosomal changes and altered protein expression, but little is known of the molecular origins of such changes. Clustered DNA damages—two or more oxidized bases, abasic sites or strand breaks on opposing strands within a few helical turns are potentially lethal and mutagenic radiation–induced damages, and could induce biological effects manifested in bystander cells. Using an insert system in which irradiated (0-60 cGy, 250 kVp X-rays) and potential bystanders share medium but are not in physical contact, we asked if unirradiated human hematopoietic 28SC cells sharing medium with the irradiated cells contained measurable levels of clustered damages. Nfo-Abasic clusters and Fpg-Oxidized Purine clusters are measured by treatment of DNA isolated from the cells with Nfo- or Fpg-proteins, pulsed-field gel electrophoresis, quantitative electronic imaging, and number average length analysis. We measured clusters levels in the irradiated and bystander cells immediately after and 24 hr after irradiation. In irradiated cells, levels of both cluster types increased linearly as a function of radiation dose. In the bystander cells, Fgp-OxyPurine clusters were induced with a dose-response similar to that of the biological bystander effects in human cells. Few, if any Nfo-Abasic clusters were detected in the bystander cells. OxyPurine clusters are induced in bystander cells to "hit" cells that were irradiated with 100 kVp X-rays. The similarity of their dose response to that of biological bystander effects suggests that clusters could be critical DNA alterations important in producing bystander changes.
Key words: bystander, DNA damage, clustered damages, human cells
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