β-L-1-[5-(E-2-Bromovinyl)-2-(Hydroxymethyl)-1,3-Dioxolan-4-yl)] Uracil (L-BHDU) Inhibits Varicella Zoster Virus Replication by Depleting the Cellular dTTP Pool

Abstractß-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) inhibits varicella zoster virus (VZV) replication in cultured cells, human skin, and in SCID-Hu mice with skin xenografts. VZV thymidine kinase converts L-BHDU to monophosphate (MP) and diphosphate (DP) forms, but the triphosphate form was not detected in infected cells and the antiviral mechanism was unknown. Given its similar structure to uridine, we asked if L-BHDU interfered with viral DNA replication via inhibition of the purine and/or pyrimidine biosynthesis pathways. Addition of purines to the medium was unable to restore VZV replication in the presence of L-BHDU. In contrast, excess thymidine and uridine in proportion to L-BHDU restored VZV replication, suggesting that the active form of L-BHDU interfered with pyrimidine biosynthesis. However, addition of thymidine and uridine failed to restore VZV replication in non-dividing cells treated with L-BHDU. Like other herpesviruses, VZV infection increased thymidine triphosphate (dTTP) in confluent cells while L-BHDU treatment decreased the dTTP pool by nearly 4-fold. The active form(s) of L-BHDU did not interfere with cellular metabolism, suggesting viral target(s).

127 THYMIDINE SYNCHRONIZATION OF IN VITRO DEVELOPMENT OF BOVINE EMBRYOS

Excess thymidine is capable of synchronizing lymphocyte cultures in vitro by acting with a feed-back mechanism during the S phase of the cell cycle (Harper 2005 Methods Mol. Biol. 296, 157–166). The possibility to synchronize the embryonic growth can be a good strategy for future epigenetic studies. The present study was undertaken to test whether excess thymidine could also synchronize in vitro development of bovine embryos. Abattoir-derived cumulus–oocyte complexes (COC) of the Agerolese breed of cattle were matured in vitro using standard procedures. After maturation, COCs were transferred in drops of 300 µL of IVF-TALP (25/drop) and covered with mineral oil. Frozen sperm from a bull were selected by centrifugation on a Percoll discontinuous gradient (45 to 80%). The pellet was diluted in IVF medium and added to the COC at the concentration of 1 × 106 sperm mL–1. After 18–20 h of gametes co-incubation, presumptive zygotes were denuded and cultured in SOF medium containing different concentrations of thymidine (0, 300, 600, 1200, 2400 µg mL–1, final concentrations). The day after (Day 2) presumptive zygotes were washed four times in fresh SOF, classified morphologically under a stereomicroscope as not divided (n.d.), 2 cells, 3–8 cells, and 9–32 cells and cultured in standard SOF at 39°C in a humidified mixture of 5% CO2,7% O2, and 88% N2. On Day 3, the embryos were again examined for the growth stage in relation to the synchronization effects. On Day 7, the embryos were evaluated for the final growth efficiency (cleavage stage and blastocyst formation). The experiment was replicated 4 times (except the 2400 µg mL–1 condition, which was replicated 2 times because of its clear toxic effect). Data were analyzed by ANOVA test. At Day 2, there were no differences between groups whatever the concentration (n.d.–2 cells: 59.7 ± 11.2, 52.9 ± 26.3, 56.1 ± 14.6, 66.0 ± 2.8, 38.0 ± 11.3; 3–8 cells : 40.3 ± 11.2, 47.1 ± 26.3, 43.9 ± 14.6, 34.0 ± 2.8, 59.9 ± 9.5; respectively for 300, 600, 1200, 2400, and control), while differences at Day 3 and 7 are shown in Table 1. The dosage of 300 µg mL–1 slowed embryo development without altering the developmental rate, whereas the other dosages were somewhat toxic to the zygotes, affecting the final percentage of blastocysts. Table 1.State of development of zygotes on Day 3 and Day 7 (cleavage and embryo rate)

18F-FLT and 125I-IdUrd uptake increase in human tumour cell lines induced by the thymidylate synthase inhibitor FdUrd

Summary Aim: 5-fluoro-2′-deoxyuridine (FdUrd) depletes the endogenous 5′-deoxythymidine triphosphate (dTTP) pool. We hypothesized whether uptake of exogenous dThd analogues could be favoured through a feedback enhanced salvage pathway and studied the FdUrd effect on cellular uptake of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) and 5-125I-iodo-2′-deoxyuridine (125I-IdUrd) in different cancer cell lines in parallel. Methods: Cell uptake of 18F-FLT and 125I-IdUrd was studied in 2 human breast, 2 colon cancer and 2 glioblastoma lines. Cells were incubated with/without 1 μmol/l FdUrd for 1 h and, after washing, with 1.2 MBq 18F-FLT or 125I-IdUrd for 0.3 to 2 h. Cell bound 18F-FLT and 125I-IdUrd was counted and expressed in % incubated activity (%IA). Kinetics of 18F-FLT cell uptake and release were studied with/without FdUrd modulation. 2′-3H-methyl-fluorothymidine (2′-3H-FLT) uptake with/without FdUrd pretreatment was tested on U87 spheroids and monolayer cells. Results: Basal uptake at 2 h of 18F-FLT and 125I-IdUrd was in the range of 0.8–1.0 and 0.4–0.6 Bq/cell, respectively. FdUrd pretreatment enhanced 18F-FLT and 125I-IdUrd uptake 1.2–2.1 and 1.7–4.4 fold, respectively, while co-incubation with excess thymidine abrogated all 18F-FLT uptake. FdUrd enhanced 18F-FLT cellular inflow in 2 breast cancer lines by factors of 1.8 and 1.6, respectively, while outflow persisted at a slightly lower rate. 2′-3H-FLT basal uptake was very low while uptake increase after FdUrd was similar in U87 monolayer cells and spheroids. Conclusions: Basal uptake of 18F-FLT was frequently higher than that of 125I-IdUrd but FdUrd induced uptake enhancement was stronger for 125I-IdUrd in five of six cell lines. 18F-FLT outflow from cells might be an explanation for the observed difference with 125I-IdUrd.

Chk1 and p21 Cooperate to Prevent Apoptosis during DNA Replication Fork Stress

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.