Association of nuclear membrane protein lamin B1 with necrosis and apoptosis in cell death induced by 5-fluoro-2'-deoxyuridine

We report that anticancer 5-fluoro-2 '-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells was performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Thus, the swelling feature for the necrosis was no longer observable, and instead cell shrinkage typical of apoptosis took place in almost all the cells examined. This finding suggests a new role for lamin B1 as a regulator in cell death.     

Catabolism of 5-fluoro-2'-deoxyuridine by isolated rat intestinal epithelial cells

The kinetics of conversion of 5-fluoro-2'-deoxyuridine (FdUrd) to 5-fluorouracil (FUra) by isolated rat intestinal epithelial cells was investigated. Also, the effects of potential inhibitors of this reaction, which is catalyzed by uridine phosphorylase and thymidine phosphorylase, were determined. A 2.5% suspension of isolated cells was incubated with FdUrd or FUra, and at specific times cells were lysed with perchloric acid and fluoropyrimidines were determined by high-performance liquid chromatography. During a 25-min incubation with either FdUrd or FUra, the amount of drug in the incubation system (total volume 0.8 ml) fell by less than 5%. However, in the presence of FdUrd, the amount of FUra increased linearly over 25 min. The apparent Vmax and Km for FUra formation were 17-27 nmole/mg DNA/min and 1.6-2.5 mM, respectively. With each nucleoside phosphorylase inhibitor, the apparent Km increased but Vmax was unaffected. The apparent Ki values were as follows (in mM): 5-nitrouracil (an inhibitor of both uridine phosphorylase and thymidine phosphorylase), 0.12; 4-thiothymine (a uridine phosphorylase-selective inhibitor), 1.52; and 6-benzyl-2-thiouracil (a thymidine phosphorylase-selective inhibitor), 0.73. It was concluded that intestinal epithelial cells are capable of degrading FdUrd to FUra and that the cells possess both uridine phosphorylase and thymidine phosphorylase activity.     

Glycosidic bond cleavage of 5-fluoro-2'-deoxyuridine and 5-fluorouridine by amino acyl-tRNA synthetases

Lysyl-tRNA synthetase isolated from rat liver cleaves glycosidic bond of 5-Fluorouridine and 5-Fluoro-2'-deoxyuridine to generate 5-Fluorouracil. The generation of 5-Fluorouracil was monitored by cellulose thin layer chromatography and by spectrophotometry. The enzyme was found to be 1.4 fold more efficient in cleaving the glycosidic bond of 5-Fluoro-2'-deoxyuridine than 5-Fluorouridine.     

Dose-dependent elimination of 5-fluoro-2'-deoxyuridine in the monkey

The kinetics of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluorouracil (FUra) disposition after bolus intravenous injection were determined in anesthetized rhesus and cynomolgus monkeys. FdUrd disappearance from plasma was an apparent triexponential process with average half-lives of 0.5, 2, and 8 min; FUra disappearance was biphasic with average half-lives of 2 and 13 min. After FdUrd injection, FUra reached peak plasma concentrations of 15-30% of the initial FdUrd concentrations within 3 min, and then disappeared more slowly than FdUrd. Total FdUrd clearance fell from 105 to 73 to 56 ml/kg/min as the dose increased from 10 to 20 to 40 mg/kg. Metabolic clearance was about 85% of total clearance and fell similarly with increasing dosage. Total and metabolic FUra clearances were about 30% of FdUrd values at an equimolar dose. Renal FdUrd clearance exceeded glomerular filtration rate and was decreased by probenecid, indicating tubular secretion; renal FUra clearance was close to glomerular filtration rate. There was no apparent correlation between dose and renal clearance or volume of distribution. It was concluded that FdUrd, like FUra, is eliminated primarily by a dose-dependent process. The metabolic basis of the dose-dependent kinetics remains to be determined.     

Synthesis and study of cyclic pronucleotides of 5-fluoro-2'-deoxyuridine

A one-step method for the synthesis of cyclic pronucleotide (cProTide) derivatives of 5-fluoro-2'-deoxyuridine (FdUrd), utilizing a novel phosphoramidating reagent, is described. Stereochemistry at phosphorus was established by NMR studies and modeling. Cytotoxicity data of representative cProTide derivatives of FdUrd are presented. The observed cell-to-cell variations in activity suggests that it is feasible to screen for structural variations in the cProTide moiety favoring metabolic activation in cancer cells, which may lead to an increase in the therapeutic effectiveness of FdUrd. The method described is applicable to all anticancer and antiviral nucleoside analogs having both the 5'- and the 3'-OH groups available for modification, forming cProTide derivatives capable of delivering the 5'-monophosphates to cells.     

Biology of 5-fluoro-2'-deoxyuridine sensitivity of Drosophila melanogaster larvae

Using axenically cultured Drosophila melanogaster, grown on defined medium, the lethal effect of 5-fluoro-2′-deoxyuridine (FUdR) at a concentration of 10^?6 M has been ascribed to inhibition of the enzyme thymidylate synthetase; in the presence of dietary thymidine the lethal dose of FUdR is increased 100-fold. Mortality under these higher concentrations is probably due to the effect of FUdR upon RNA synthesis. Larval transfer between media with and without FUdR indicates a prolonged period during larval life when the base analogue is effective, both under conditions where suppression of DNA and RNA synthesis seem to cause death.The response of fruit-fly larvae to FUdR probably reflects the similarity of deoxyribopyrimidine metabolism in Drosophila to that in other organisms. However, finding that deoxycytidine is almost as efficient as thymidine in relieving the killing effect of low concentrations of FUdR suggests that some aspects of nucleotide metabolism in Drosophila remain to be elucidated.     

Measurement of 5-fluoro-2'-deoxyuridine serum levels by gas chromatography chemical ionization mass spectrometry

Serum levels of 5-fluoro-2'-deoxyuridine in cancer treated patients were measured by gas chromatography mass spectrometry under chemical ionization conditions; 1-(2-deoxy-beta-D-lyxofuranosyl)-5-fluorouracil (3'-epi-5-fluoro-2'-deoxyuridine) was used as an internal standard. The drug and internal standard were quantitatively isolated from the serum sample by a mini-column anion exchange method and the extract permethylated using potassium-tert-butoxide in dimethylsulphoxide and methyl iodide. The derivatized nucleosides were then re-extracted from the reaction mixture and analysed on a glass capillary column coated with Superox-4. The column was coupled directly to the chemical ionization source of the mass spectrometer; NH3 was used as the reagent gas. The gas chromatographic effluent was monitored at m/z 289, the [MH]+ ion of the N,O-permethyl derivatives of 5-fluoro-2'-deoxyuridine and the internal standard. Recovery of 5-fluoro-2'-deoxyuridine from serum in the 0-1 microgram ml-1 concentration range averaged 93 +/- 2% (SD); a linear detector response was observed up to 50 ng 5-fluoro-2'-deoxyuridine ml-1. At the 10 ng ml-1 level, a within-run assay precision of 10% (CV) (n = 5) was found, while a detection limit of about 1 ng 5-fluoro-2'-deoxyuridine ml-1 of serum was attained. The method was applied to the measurement of disappearance curves of 5-fluoro-2'-deoxyuridine in the serum of treated patients.     

Interaction of thymidylate synthase with the 5'-thiophosphates, 5'-dithiophosphates, 5'-H-phosphonates and 5'-S-thiosulfates of 2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine.

New analogs of dUMP, dTMP and 5-fluoro-dUMP, including the corresponding 5'-thiophosphates (dUMPS, dTMPS and FdUMPS), 5'-dithiophosphates (dUMPS2, dTMPS2 and FdUMPS2), 5'-H-phosphonates (dUMP-H, dTMP-H and FdUMP-H) and 5'-S-thiosulfates (dUSSO3, dTSSO3 and FdUSSO3), have been synthesized and their interactions studied with highly purified mammalian thymidylate synthase. dUMPS and dUMPS2 proved to be good substrates, and dTMPS and dTMPS2 classic competitive inhibitors, only slightly weaker than dTMP. Their 5-fluoro congeners behaved as potent, slow-binding inhibitors. By contrast, the corresponding 5'-H-phosphonates and 5'-S-thiosulfates displayed weak activities, only FdUMP-H and FdUSSO3 exhibiting significant interactions with the enzyme, as weak competitive slow-binding inhibitors versus dUMR The pH-dependence of enzyme time-independent inhibition by FdUMP and FdUMPS was found to correlate with the difference in pKa values of the phosphate and thiophosphate groups, the profile of FdUMPS being shifted (approximately 1 pH unit) toward lower pH values, so that binding of dUMP and its analogs is limited by the phosphate secondary hydroxyl ionization. Hence, together with the effects of 5'-H-phosphonate and 5'-S-thiosulfate substituents, the much weaker interactions of the nucleotide analogs (3-5 orders of magnitude lower than for the parent 5'-phosphates) with the enzyme is further evidence that the enzyme's active center prefers the dianionic phosphate group for optimum binding.     

Assay of intracellular thymidylate synthetase activity and inhibition by 5-fluoro-2'-deoxyuridine in lymphocytes.

Thymidylate synthetase catalyses the formation of thymidine monophosphate from deoxyuridine monophosphate. Purified thymidylate synthetase can be assayed radiochemically using labelled deoxyuridine monophosphate as substrate, but cells are impervious to deoxyuridine monophosphate and so intracellular thymidylate synthetase activity cannot be assayed in this way. In this paper we describe the assay of intracellular thymidylate synthetase activity in intact cells using labelled 2'-deoxyuridine. The assay showed linear kinetics with respect to time, concentration of 2'-deoxyuridine, and cell concentration. 5-fluoro-2'-deoxyuridine inhibited intracellular thymidylate synthetase activity measured with this assay by 50% at 5 nM. Cell growth was inhibited by 50% at 6 nM 5-fluoro-2'-deoxyuridine. The assay was specific for thymidylate synthetase and enabled measurement of thymidylate synthetase activity in situ in intact cells.     

Radiotoxicity of 5-[125I]iodo-2'-deoxyuridine in mammalian cells following treatment with 5-fluoro-2'-deoxyuridine.

We have enhanced the uptake of 5-[125I]iodo-2'-deoxyuridine (125IUdR) in Chinese hamster V79 cells with 5-fluoro-2'-deoxyuridine (FUdR) and have examined the combined toxicity of these agents. Although the uptake of 125IUdR increases approximately 3.2 +/- 0.5-fold in the presence of 1 microM FUdR, when cell survival fraction is plotted as a function of intranuclear 125IUdR content, the biphasic curve obtained reaches a plateau at a higher survival fraction than with control cells not exposed to FUdR. The results suggest that a greater number of cells were prevented from entering the S phase and consequently from incorporating 125IUdR. An FUdR- 125IUdR combination, therefore, does not seem to enhance the therapeutic potential of 125IUdR. Such observations are also of importance when FUdR and other inhibitors are used to enhance cold IUdR uptake in an effort to obtain an increase in radiosensitization effects.     

Protein expression profiles in Saccharomyces cerevisiae during apoptosis induced by H2O2

We identified the proteins involved during apoptosis induced by H2O2 in Saccharomyces cerevisiae, and analyzed the global protein pattern by 2-DE. We analyzed classical parameters of apoptosis such as chromatin condensation, DNA fragmentation, and morphology changes of cells. Exposure of yeast cells to nonphysiological doses of peroxides decreases the expression (or increases degradation) of enzymes involved in protection against oxidative stress. This leads the yeast cells to a reduction of their antioxidant defense and makes the cells more prone to apoptosis. In our data the down expression of peroxiredoxin II and GST I, could induce a perturbation of mitochondrial function with an alteration of permeability of the membrane leading to the mitochondria-mediated apoptosis. Moreover, we identified a new spot of a classical glycolytic enzyme: the glyceraldehyde 3-phosphate dehydrogenase during apoptosis. It is known that GAPDH is an extremely abundant glycolytic enzyme with multiple functions and that its overexpression is evident during apoptosis induced by a variety of stimuli. Our results confirm that it is a major intracellular messenger mediating apoptotic death and that this new spot of GAPDH could be an intracellular sensor of oxidative stress during apoptosis induced by H2O2 in S. cerevisiae.     

Collateral resistance of a dideoxycytidine-resistant cell line to 5-fluoro-2'-deoxyuridine.

Exposure of a human lymphocytic cell line, H9 cells, to 0.5 microM and 5.0 microM dideoxycytidine (ddC) resulted in isolation of ddC-resistant H9-ddC0.5w and H9-ddC5.0w cell lines. In addition, these cell lines were also resistant to azidothymidine and had reduced deoxycytidine kinase and thymidine kinase activities. We now show that these cell lines are 4-fold and 2000-fold collaterally resistant to 5-fluoro-2'-deoxyuridine (FdUR), respectively, but not to 5-fluorouracil (FU). Biochemical evaluations show that, compared to the parental cells, the FdUR phosphorylation was reduced to 36.3% and 9.2% and the FdUMP levels were decreased to 48.1% and 1.2% in these cell lines. Taken together, the data suggest that ddC, an antiviral agent, is capable of inducing resistance to FdUR-a drug that is not its analog and which has a different metabolism, target site, and mechanism of action.     

A sensitive method for determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in human plasma by high-pressure liquid chromatography

Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.     

Effects of 5-fluoro-2'-deoxyuridine on cell proliferation in the developing mouse palate

Cell proliferation following the administration of 5-fluoro-2'-deoxyuridine (FUdR) to pregnant mice was studied in the vertical palatal processes of fetuses using 3H-thymidine and autoradiography. Doses of 30 and 80 mg FUdR per kilogram maternal body weight substantially reduced the number of labelled nuclei in the epithelium and mesenchyme. Both doses of FUdR also decreased the mean number of silver grains over mesenchymal nuclei. It is concluded that an important mechanism, whereby FUdR induces cleft palate in experimental animals, is by reducing cell proliferation and the rate of DNA synthesis in the palatal processes.     

MicroRNA-351 Regulates Two-Types of Cell Death,Necrosis and Apoptosis,Induced by 5-fluoro-2'-deoxyuridine

Cell-death can be necrosis and apoptosis. We are investigating the mechanisms regulating the cell death that occurs on treatment of mouse cancer cell-line FM3A with antitumor 5-fluoro-2''-deoxyuridine (FUdR): necrosis occurs for the original clone F28-7, and apoptosis for its variant F28-7-A. Here we report that a microRNA (miR-351) regulates the cell death pattern. The miR-351 is expressed strongly in F28-7-A but only weakly in F28-7. Induction of a higher expression of miR-351 in F28-7 by transfecting an miRNA mimic into F28-7 resulted in a change of the death mode; necrosis to apoptosis. Furthermore, transfection of an miR-351 inhibitor into F28-7-A resulted in the morphology change, apoptosis to necrosis, in this death-by-FUdR. Possible mechanism involving lamin B1 in this miR-351’s regulatory action is discussed.     

5-Fluorouracil derivatives. III. A simple method to prepare 5-fluoro-2'-deoxyuridine derivatives

3',5'-Diacyl-2'-bromo-5-fluoro-2'-deoxyuridine (4) was obtained by the reaction of 5, 6-dihydro-6-hydroxy-5-fluorouridine (2) and acyl bromide. Because the route from uridine (1) to 2, the route from 4 to 3',5'-diacyl-5-fluoro-2'-deoxyuridine (5), and the route from 5 to 5-fluoro-2'-deoxyuridine (FUDR, 6) are known reactions, the three step synthesis from uridine to 5 and four step synthesis from uridine to FUDR have been accomplished.