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) ha ve focused on the molecular mechanisms underlying regulation of the cell cy cle, 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 rat io, which subsequently results in enhanced DNA fragmentation and cell death . To better understand the mechanism of FP-RS, me characterized the cellula r and biochemical responses to ionizing radiation (IR) alone, using differe nt synchronization techniques in two isogenic, TS-deficient mutant cell lin es, JH-1 (TS-) and JH-2 (Thy4), derived previously from a human colon cance r cell line. After G(0) synchronization by leucine deprivation, these clone s 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 deat h (<24 h). No difference in the late S-phase and G(2)-M cell populations we re noted between these growth-stimulated, G(0)-synchronized TS-deficient ce ll lines with dThd withdrawal. Biochemically, the intracellular ratio of dA TP: dTTP increased substantially in JH-l cells as cells progressed into ear ly S-phase compared with JH-2 cells, which remained in G(1) phase. Synchron ized JH-l cells showed significantly decreased clonogenic survival and an i ncrease 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 qu adratic 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 ce lls, synchronized in late G(1) and following growth stimulation, now progre ssed into S-phase identically (<24 h), with similarly increased dATP:dTTP r atios under dThd withdrawal conditions. These late G(1)-synchronized JH-l a nd 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 t he G(1) restriction point under an altered (increased) dATP:dTTP ratio is a major determinant of FP-RS.