title: Radiosensitivity of thymidylate synthase-deficient human tumor cells is affected by progression through the G1 restriction point into S-phase: implications for fluoropyrimidine radiosensitization.
abstract: 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 alpha and decrease in beta, 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.
authors.affiliation: Department of Radiation Oncology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, USA.
authors.forename: H S
authors.initials: HS
authors.lastname: Hwang
authors.forename: T W
authors.initials: TW
authors.lastname: Davis
authors.forename: J A
authors.initials: JA
authors.lastname: Houghton
authors.forename: T J
authors.initials: TJ
authors.lastname: Kinsella
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017209:Apoptosis; D002470:Cell Survival; D004249:DNA Damage; D003853:Deoxyribonucleosides / Q000378:metabolism* / Q000494:pharmacology; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects*; D006801:Humans; D009363:Neoplasm Proteins / Q000172:deficiency*; D011743:Pyrimidines; D011836:Radiation Tolerance; D016192:Resting Phase, Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects*; D016196:S Phase / Q000187:drug effects / Q000528:radiation effects*; D013940:Thymidylate Synthase / Q000172:deficiency*; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D003853:Deoxyribonucleosides; D009363:Neoplasm Proteins; D011743:Pyrimidines; D013942:Thymine Nucleotides; C029269:5-fluoropyrimidine; D000255:Adenosine Triphosphate; D013940:Thymidylate Synthase; C024157:thymidine 5'-triphosphate