RESPONSE OF BROMODEOXYURIDINE-SUBSTITUTED CHINESE-HAMSTER CELLS TO UVA LIGHT EXPOSURE IN THE PRESENCE OF HOECHST DYE NUMBER-33258 - SURVIVAL AND DNA-REPAIR STUDIES

Previous work has established that DNA double-strand breaks (DSBs) are formed when Chinese hamster cells are substituted with 5-bromo-2'-deo xyuridine (BrdU) and exposed to UVA light in the presence of Hoechst d ye #33258. Double-strand breaks produced by this treatment (5.1 x 10(- 6) DSBs/BrdU residue/kJ m(-2)) were found to depend linearly on the le vel of BrdU substitution, Hoechst dye and fluence of UVA light. To exa mine the biological consequences of these novel DSBs, clonogenic assay s were used to score cell survival, and elution assays were used to me asure strand break levels at various times after photolysis. Using thi s system, marked cell killing was observed; photosensitivity could be increased by four orders of magnitude compared to cells without BrdU a nd dye. Decreases in the Fo value and the shoulder of survival curves followed increasing levels of BrdU substitution. In addition, the resu lts indicate that DSBs produced by this photolysis protocol are two to three times more effective in causing cell killing than the DSBs prod uced by the action of ionizing radiation. To investigate the cause of the toxicity, repair of DSBs after photolysis was measured. Unexpected ly, DSB levels increased two- to threefold over 1 h at 37 degrees C, t hen decreased to initial damage levels over the next 2 h. The implicat ions of this break induction are discussed in terms of mechanism and c ell killing.