The long term objective of this proposal is the design of a rapid predictive assay of tumor radiosensitivity to assist in the design of patient-specific treatments. For example, radioresistant tumors may be best controlled by either higher total doses of radiation or alternative treatment modalities. It has been found that after cellular irradiation, supercoiled DNA from relatively radiosensitive cells is only poorly rewound by the DNA intercalator ethidium bromide. This can be measured within a flow cytometer by monitoring the light scattered from nucleoids extracted from single cells. It is proposed that the reduced ability of DNA supercoils from irradiated radiosensitive cells to maintain supercoil tension is due to increased relative movement between the base of each DNA loop and its coordinating site at the nuclear matrix. This weakness in DNA association with the matrix allows the propagation of loop to loop supercoil relaxation, by the rotation of matrix DNA around its own long axis, driven by the energy released as a damaged supercoiled loop unwinds. It is further proposed that differences in DNA matrix stability are mediated through the structural or enzymatic activity of DNA topoisomerase II. Such a change in structural stability may affect the effectiveness of DNA repair leading to the difference in radiosensitivity seen. This is to be tested by: SITE-SPECIFICITY OF DNA BREAKS AND THEIR REPAIR IN DISTAL vs MATRIX ASSOCIATED DNA. The induction and repair of single and double stranded DNA breaks will be measured, using alkaline elution and pulse field gel methods, analyzing both DNA that is distal to the replication origin and that which is recently synthesized and therefore more closely linked to, and affected by, the nuclear matrix. ALTERATION OF SUPERCOILING TENSION AS A MEDIATOR OF DNA-MATRIX BINDING AFFINITY. Supercoil tension will be modified using both the topoisomerase II specific inhibitors, VP16 and MAMSA, by experimentally exhausting topoisomerase II content by taking the cells out of cycle, and after nucleoid extraction by ethidium bromide intercalation. The effect of these treatments on DNA-nuclear matrix binding will be determined directly, by restriction enzyme digestion of accessible DNA, and by the ability to maintain positive supercoiling as assayed by nucleoid flow cytometry. ROLE OF TOPOISOMERASE II IN MEDIATING CHANGES IN RADIOSENSITIVITY. The amount and activity of DNA topoisomerase II that is a component of the nuclear matrix will be measured by both specific antisera and its ability to unknott plasmid DNA. Variations in structure or activity of the enzyme may provide a biochemical explanation underlying differences in DNA repair and nuclear supercoiling behavior expected from the execution of Aims 1 and 2.
| Hjelle, B; Mills, R; Goldsmith, C et al. (1992) Primary isolation of human T-cell leukemia-lymphoma virus types I and II: use for confirming infection in seroindeterminate blood donors. J Clin Microbiol 30:2195-9 |
