Potentially lethal damage (PLD) is an operationally defined section of radiation-induced cellular damage whose expression is modifiable by post-treatment environmental conditions. Agents currently known to affect the expression of PLD following ionizing radiation include methylated xanthines, purine nucleoside analogs, nicotinamide analogs, intercalating antibiotics and anisotonic solutions. The objective of the proposed study is to test the hypothesis that chromatin structure is a determining factor in the repair of PLD and its corollary that altering chromatin structure should modify the response of human cell lines to DNA damaging agents. To the extent that our hypothesis is correct, exploiting chromatin structural differences may provide a selective strategy for modifying the expression of PLD in human cells in a manner that is dependent upon proliferative activity or transformation state (both are related to chromatin conformation changes). This possibility is of relevance to radiotherapy, where the repair of PLD may be a major cellular determinant in the radiocurability of human tumors in multi- fraction schedules. To test the hypothesis, we will systemically examine in Specific Aim 1 the effects of 6 known modifiers of x- ray PLD expression on chromatin structure as assessed by the ability of DNA to undergo supercoiling changes, the accessibility of DNA to enzymatic digestion and differences in the structural protein components of the nucleus and nuclear matrix.
In Specific Aim 3, the opposite approach is taken. Chemical agents known to alter chromatin structure will be tested for their ability to modify PLD expression following ionizing radiation. The studies in Specific Aim 2 will address whether cell cycle dependent changes in chromatin conformation contribute to the observed (in Aims 1 and 3) effects on PLD expression.
In specific Aim 4, data indicating altered PLD expression and chromatin structural changes will be related to the repair of DNA damage at the molecular level. Finally, to demonstrate whether modifying chromatin structure can alter the radiation response of cells in a selective manner, comparative studies will be performed using cell lines differing in proliferation status, transformation state and inherent PLD repair capacity.
