The thiol-mediated modification of radiation induced DNA strand break induction and cell survival after irradiation

This dissertation addresses the mechanism by which thiol compounds can modify cellular radiation response. The differential radiation response between aerobic and hypoxic conditions is well established for the experimental endpoints of cell and DNA damage and is commonly known as the Oxygen Effect or the oxygen Enhancement Ratio (OER). intracellular non-protein thiol compounds had been presumed responsible for enhanced cell survival or reduced DNA damage after irradiation in hypoxic conditions. The use of a fluorescent thiol binding agent to quantify the dominant non-protein thiol compound glutathione (GSH) against the background of protein thiols in individual cells with flow cytometry has been done. It was found that cell line specific variability interfered with uniform GSH quantitation by flow cytometry unless a membrane permeabilization technique was employed. Measurement of GSH content of cells dissociated from 9L rodent tumors invalidated the clinical utility of this assay due to irreversible thiol depletion during the disaggregation process. The DNA damage work presented herein utilizes three different DNA damage assays: alkaline elution, neutral elution and Pulsed Field gel electrophoresis (PFGE), to demonstrate an important role for nuclear protein thiols. Isolated nuclei were prepared by two different methods wherein non-protein thiols were virtually eliminated, yet a measurable OER was still evident. Under these circumstances, it was possible to conclude that nuclear protein thiols play a much more important role in the production of the OER than had been suspected. Further work to identify the principle proteins involved in this process is planned. Regarding cell survival after irradiation, this dissertation utilizes a novel technique of cell suspension culture wherein human colon tumor (HCT-116 line) cells were irradiated at 37°C with continuous monitoring of oxygen tension and very accurate intracellular thiol measurements combined with traditional survival techniques to reveal an unexpectedly significant role for cysteine as a cellular radiation protector. This work strongly supports the clinical significance of high cysteine content in rodent tumors by this laboratory.