THE ROLE OF FLUDARABINE-INDUCED APOPTOSIS AND CELL-CYCLE SYNCHRONIZATION IN ENHANCED MURINE TUMOR RADIATION RESPONSE IN-VIVO

We have previously reported that fludarabine, an adenine nucleoside an alogue, significantly enhances radiation-induced tumor regrowth delay and Local cure in several. mouse tumors. Although fludarabine potentia ted tumor regrowth delay at various times from -36 h to +6 h in a SA-N H mouse sarcoma model, the greatest enhancement was observed when flud arabine was administered 24 h before irradiation. The purpose of this study was to understand the basis for in vivo enhancement of radiation efficacy by fludarabine. To examine the effect of fludarabine on DNA synthesis and cell cycle progression, tumor-bearing mice were given fl udarabine by an i.p. route and then bromodeoxyuridine at various times up to 36 h, followed 0.5 h later by tumor harvest. Two-parameter flow cytometry analysis of the tumor cells using an anti-bromodeoxyuridine antibody demonstrated that an 800-mg/kg fludarabine dose stops DNA sy nthesis within 3 h with recovery starting at 12 h. By 24 h after fluda rabine treatment, a synchronized wave of cycling tumor cells appeared in G(2)-M phase. The degree of DNA synthesis shutdown and the timing o f the reinitiation of DNA synthesis and cell cycle progression were al l fludarabine dose dependent. Interestingly, DNA synthesis reinitiated only at the G(1)-S boundary; tells in the S phase at the time of flud arabine administration appeared to disappear from the tumor population . To confirm these observations more directly, we pretreated tumor-bea ring mice i.p. with chlorodeoxyuridine to mark the cells in the S phas e, gave them fludarabine 0.5 h later, and then gave them iododeoxyurid ine 0.5 h before tumor harvest. Mow cytometry analysis using antibodie s specific for chlorodeoxyuridine- and iododeoxyuridined-labeled cells confirmed that cells in the S phase at the time of fludarabine admini stration never reinitiated DNA synthesis and disappeared from the tumo r population. Immunohistological analysis of tumor sections obtained a fter fludarabine administration demonstrated that prelabeled S-phase c ells took on an apoptotic appearance and gradually disappeared from th e tumors. An in situ DNA end labeling assay demonstrated DNA fragmenta tion in these morphologically apoptotic cells. These results suggest t hat the mechanism of fludarabine enhancement of radiation response inv olves induced S-phase cell loss through an apoptotic pathway and subse quent synchronization of the remaining cells to a more radiosensitive cell cycle phase at the time of irradiation,