This proposal will test the hypothesis that sulfhydryl redox status is a major determinant of the cytotoxicity of ionizing radiation and many chemotherapeutic drugs. Reducing conditions are expected to protect against cytotoxicity; oxidizing conditions should amplify damage. To test this hypothesis requires an assay of thiol redox status in several biological test systems where the intracellular thiol status can be modified in a stepwise and controlled way. A new technique has been developed in out laboratory where both thiol and disulfide forms of the same probe molecule can be detected by NMR. Either 13C or 19F can be used as reporters of the oxidation state of nearby S. New probe molecules must be synthesized where the redox concentration ratio will be close to unity. After showing that the NMR measurement of the sulfhydryl redox ratio correlates with response to therapy, a more convenient assay can be developed using 19F peak suppression NMR imaging. 19F has superior properties for detection and does not contribute to the in vivo background signal. The new thiol probes will be characterized in several ways: Eo', partition coefficients of the reduced and oxidized forms, and any interference caused by physiologic constituents, e.g. Ca++. Tumor cell lines will be grown both in culture and in basement membrane gel threads and their sulfhydryl redox status under various growth conditions will be compared to that of the same cell line grown in vivo. NMR measurements of concentration ratio will be correlated with chemical measurements of thiol and disulfide by HPLC. The central biological hypothesis of the research proposal can be tested before the optimal probe molecule is selected, using cells grown in vitro and treated with radiation or redox-altering drugs. The clonogenic survival of these cells will be measured. The hypothesis predicts that a shift in redox status toward oxidizing conditions would sensitize the cells, observable as decreased survival. In other experiments a shift toward reducing conditions should promote survival. Sulfhydryl depletion without changing redox, e.g. by formation of thioethers, should change survival only minimally. After showing a correlation between the NMR redox measurement and survival after therapy, we will extend biological validation to additional cell lines and therapy protocols to generalize the conclusions. The most appropriate redox probe will penetrate cells adequately and have the desired biodistribution and electrochemical characteristics. Interaction of the thiol redox probe with other cellular redox couples will be evaluated, to extend its potential as an in vivo redox indicator. Development of a probe molecule of appropriate Eo' and high sensitivity for detection win allow NMR interrogation of both tumor and surrounding normal tissue to discern likely outcomes of drug or radiation therapy.
| Casciari, J J; Chin, L K; Livesey, J C et al. (1995) Growth rate, labeling index, and radiation survival of cells grown in the Matrigel thread in vitro tumor model. In Vitro Cell Dev Biol Anim 31:582-9 |
