Radiosensitivity Of Thymidylate Synthase-Deficient Human Tumor Cells Is Affected By Progression Through The G(1) Restriction Point Into S-Phase: Implications For Fluoropyrimidine Radiosensitization

Recent studies of fluoropyrimidine (FP)-mediated radiosensitization (RS) have focused on the molecular mechanisms underlying regulation of the cell cycle, particularly at the G(1)-S transition. Although thymidylate synthase (TS) inhibition by FP is necessary, we hypothesize that FP-RS is temporally dependent on progression of cells into S-phase under conditions of altered deoxynucleotide triphosphate pools, particularly an increased dATP:dTTP ratio, which subsequently results in enhanced DNA fragmentation and cell death. To better understand the mechanism of FP-RS, me characterized the cellular and biochemical responses to ionizing radiation (IR) alone, using different synchronization techniques in two isogenic, TS-deficient mutant cell lines, JH-1 (TS-) and JH-2 (Thy4), derived previously from a human colon cancer cell line. After G(0) synchronization by leucine deprivation, these clones differ under subsequent growth conditions and dThd withdrawal: JH-2 cells have an intact G(1) arrest (>72 h) and delayed cell death (>96 h), whereas JH-1 cells progress rapidly into early S-phase and undergo acute cell death (<24 h). No difference in the late S-phase and G(2)-M cell populations were noted between these growth-stimulated, G(0)-synchronized TS-deficient cell lines with dThd withdrawal. Biochemically, the intracellular ratio of dATP: dTTP increased substantially in JH-l cells as cells progressed into early S-phase compared with JH-2 cells, which remained in G(1) phase. Synchronized JH-l cells showed significantly decreased clonogenic survival and an increase in DNA fragmentation after IR when compared with JH-2 cells. RS was demonstrated by an increase in alpha and decrease in beta, using linear quadratic analyses. An alternative synchronization technique used mimosine to induce a block in late G(1), close to G(1)-S border. Both JH-1 and JH-2 cells, synchronized in late G(1) and following growth stimulation, now progressed into S-phase identically (<24 h), with similarly increased dATP:dTTP ratios under dThd withdrawal conditions. These late G(1)-synchronized JH-l and JH-2 cells also showed a comparable reduction in clonogenic survival and similar patterns of increased DNA fragmentation following IR, We suggest, based on the cellular and biochemical differences in response to IR between G(0)- and late G(1)-synchronized cells, that S-phase progression through the G(1) restriction point under an altered (increased) dATP:dTTP ratio is a major determinant of FP-RS.