The aims of this proposal are to: (1) test the hypothesis that radiation-induced division delay is not caused by damage to DNA, but is caused by damage to a non-DNA structure(s) associated with DNA at specific times in the cell cycle; (2) determine the cell cycle time of association; and (3) establish the relationship between damage to this non-DNA structure (i.e., division delay) and cell mortality. To accomplish these aims, Chinese hamster ovary (CHO) cells will be pulse labeled with 125-IUdR so that 125-I decays will irradiate DNA and its closely associated structures, but only inhibit cell progression in a portion of the labeled cells. 125-I decays will be specifically accumulated at various times in the cell cycle, by cooling to prevent progression. To avoid dependence on a single technique, two methods will be used to measure the effect of differential, cell cycle accumulation of 125-I decays on division delay: (1) 125-IUdR labeled cells will be selected in mitosis and decays accumulated at points throughout the cell cycle; (2) after 125-IUdR labeling, decays will be accumulated while the cells are either only in S- or in S- plus G2-phases, before they reach the first mitosis. In each case, the effect 125-I decays on cell entry into the subsequent mitosis will be assessed by comparing the time and rate of entry with and without accumulation of decays. The cell cycle association between DNA and the division delay structure can also be determined from these results; accumulation of 125-I decays will only affect cell progression at the times that the DNA and the non-DNA structure are associated. The relationship between 125-I-induced division delay and cell mortality will be based on the differences between the survival curves of the delayed and non-delayed populations. The long range goals of this research is to use the ability to separate 125-I-induced division delayed and non-delayed cells to identify and then study a cell-survival-related, non-DNA structure. Ultimately, identification of this structure could lead to new approaches to cancer therapy that depend, in part, on interaction with a non-DNA target.
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