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Radiosensitivity of Thymidylate Synthase-deficient Human Tumor Cells Is Affected by Progression through the G₁ Restriction Point into S-Phase: Implications for Fluoropyrimidine Radiosensitization¹

Department of Radiation Oncology, Case Western Reserve University, School of Medicine and University Hospitals of Cleveland/Ireland Cancer Center, Cleveland, Ohio 44106 [H-S. H., T. W. D., T. J. K.], and Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38101 [J. A. H.]

Recent studies of fluoropyrimidine (FP)-mediated radiosensitization (RS) have focused on the molecular mechanisms underlying regulation of the cell cycle, particularly at the G₁-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, we 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 G₀ synchronization by leucine deprivation, these clones differ under subsequent growth conditions and dThd withdrawal: JH-2 cells have an intact G₁ 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 G₂-M cell populations were noted between these growth-stimulated, G₀-synchronized TS-deficient cell lines with dThd withdrawal. Biochemically, the intracellular ratio of dATP:dTTP increased substantially in JH-1 cells as cells progressed into early S-phase compared with JH-2 cells, which remained in G₁ phase. Synchronized JH-1 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 and decrease in ß, using linear quadratic analyses. An alternative synchronization technique used mimosine to induce a block in late G₁, close to G₁-S border. Both JH-1 and JH-2 cells, synchronized in late G₁ and following growth stimulation, now progressed into S-phase identically (<24 h), with similarly increased dATP:dTTP ratios under dThd withdrawal conditions. These late G₁-synchronized JH-1 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 G₀- and late G₁-synchronized cells, that S-phase progression through the G₁ restriction point under an altered (increased) dATP:dTTP ratio is a major determinant of FP-RS.

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