THE ROLE OF CELL-CYCLE REDISTRIBUTION IN RADIOSENSITIZATION - IMPLICATIONS REGARDING THE MECHANISM OF FLUORODEOXYURIDINE RADIOSENSITIZATION

Purpose: Radiosensitization has previously been demonstrated in a huma n colon cancer cell line (HT-29) following a 2 h exposure to low, clin ically relevant concentrations (0.05-0.5 mu M) of fluorodeoxyuridine ( FdUrd) (15). The sensitizer enhancement ratio value (measured at 10% s urvival) plateaued at approximate to 1.7 between 16 and 32 h following removal of drug. Parallel studies investigating the effect of FdUrd o n the distribution of cells throughout the cell cycle found that the p ercentage of cells in early S-phase increased to approximate to 70% du ring the same period that maximal radiosensitization was noted. As a f ollow-up to these findings, experiments have been designed to investig ate the contribution of this early S-phase delay to radiosensitization . Methods and Materials: Synchronized populations of HT-29 cells have been obtained with three separate techniques. Two involve the inductio n of a reversible metaphase arrest (with high pressure N2O or colcemid ) followed by a shakeoff of mitotic cells. The third uses a plant amin o acid, mimosine, to induce a reversible block at the G(1)/S boundary. Flow cytometry was used to analyze the degree of synchrony based on b romodeoxyuridine (BrdUrd) uptake and propidium iodide (PI) staining. R adiation survival curves were obtained on these synchronized populatio ns to investigate changes in radiosensitivity through the cell cycle. Additionally, levels of thymidylate synthase (TS), the primary target of FdUrd cytotoxicity, were measured in each phase of the cell cycle u sing the TS 106 monoclonal antibody against human TS. Results: Synchro nization with mitotic shakeoff produced relatively pure populations of cells in G(1); however, the degree of synchrony in early S-phase was limited both by cells remaining in GI and by cells progressing into la te S-phase. These techniques failed to reveal increased radiosensitivi ty in early S-phase at 10% survival. An 18 h exposure to mimosine resu lted in populations that more closely resembled the early S-phase enri chment following FdUrd exposure and revealed increased radiosensitivit y during early S-phase. TS levels were noted to be only 1.3 times high er in S phase than in G(0)/G(1). Conclusion: Radiation survival data f rom cells synchronized with mitotic shakeoff techniques suggest that e arly S-phase delay is unlikely to be the primary mechansim of FdUrd ra diosensitizatian. In contrast, the increased sensitivity seen in early S-phase with mimosine synchronized cells is similar to that seen with FdUrd. Although confounding biochemical pertubations cannot be ruled out, these data continue to suggest an association between early S-pha se enrichment and radiosensitization. The significance of TS inhibitio n as a mechanism of FdUrd radiosensitization remains unclear.