Methods for the complete analysis of 5-fluorouracil metabolites in cell extracts

Pogolotti, Alfonso L.; Nolan, Patricia A.; Santi, Daniel V.

doi:10.1016/0003-2697(81)90708-9

Cited by 50 publications

(20 citation statements)

References 17 publications

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“…The acid supernatant was recovered and added to an equal volume of cold freon containing 0.5 M tri-n-octylamine. The mixture was vortexed for 15 s and centrifuged for 30 s at 12,000 ϫ g. The aqueous upper phase was recovered and analyzed by high performance liquid chromatography, as described previously (28). Thymidine metabolite peaks from cell extracts were identified by comparison of retention times using thymidine mono-, di-, and triphosphates as standards.…”

Section: Analysis Of Intracellular Tritium-labeled Thymidine Metabolimentioning

confidence: 99%

p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

Magro

Russo

et al. 2004

Cancer Research

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The p14 ARF protein, the product of an alternate reading frame of the INK4A/ARF locus on human chromosome 9p21, disrupts the ability of MDM2 to target p53 for proteosomal degradation and causes an increase in steady-state p53 levels, leading to a G 1 and G 2 arrest of cells in the cell cycle. Although much is known about the function of p14 ARF in the p53 pathway, not as much is known about its function in human tumor growth and chemosensitivity independently of up-regulation of p53 protein levels. To learn more about its effect on cellular proliferation and chemoresistance independent of p53 up-regulation, human HT-1080 fibrosarcoma cells null for p14 ARF and harboring a defective p53 pathway were stably transfected with p14 ARF cDNA under the tight control of a doxycyclineinducible promoter. Induction of p14 ARF caused a decrease in cell proliferation rate and colony formation and a marked decrease in the level of dihydrofolate reductase (DHFR) protein. The effect of p14 ARF on DHFR protein levels was specific, because thymidylate kinase and thymidylate synthase protein levels were not decreased nor were p53 or p21WAF1 protein levels increased. The decrease in DHFR protein was abolished when the cells were treated with the proteasome inhibitor MG132, demonstrating that p14 ARF augments proteasomal degradation of the protein. Surprisingly, induction of p14ARF increased resistance to the folate antagonists methotrexate, trimetrexate, and raltitrexed. Depletion of thymidine in the medium reversed this resistance, indicating that p14 ARF induction increases the reliance of these cells on thymidine salvage.

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Section: Analysis Of Intracellular Tritium-labeled Thymidine Metabolimentioning

confidence: 99%

p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

Magro

Russo

et al. 2004

Cancer Research

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“…Ribonucleoside mono-, di-and triphosphates were separated at a flow rate of 2 ml/min by isocratic elution at 7 mM ammonium phosphate, pH 3.8, for 9 min, followed by a 45-min linear gradient to 0.25 M ammonium phosphate, 0.5 M KCl, pH 4.5, and a final 15 min linear elution with the latter buffer [31]. When only nucleoside triphosphates were to be measured, an isocratic elution at 0.4 M ammonium phosphate, 2.5% acetonitrile, pH 3.25, was used.…”

Section: Measurement Of Intracellular Nucleotide Concentrationsmentioning

confidence: 99%

On the mechanism of deoxyribonucleoside toxicity in human T-lymphoblastoid cells. Reversal of growth inhibition by addition of cytidine

Dahbo

Eriksson

1985

Eur J Biochem

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High levels of deoxyadenosine and deoxyguanosine in patients with inherited deficiency of either adenosine deaminase or purine-nucleoside phosphorylase, respectively, are considered to be responsible for the associated immunological disorder. The mechanism involves phosphorylation to the corresponding deoxyribonucleoside triphosphates which subsequently inhibit the CDP-reducing activity of ribonucleotide reductase. Addition of deoxycytidine protects cells from the cytotoxic effects of deoxyadenosine and deoxyguanosine by competition for phosphorylation and by replenishing dCTP, the apparent limiting DNA precursor. Addition of cytidine, but not uridine, led to a reversal of deoxyguanosine and thymidine growth inhibition, comparable to that obtained with deoxycytidine. Analysis of the intracellular nucleotide pools showed that increased levels of cytidine ribonucleotides were sufficient to overcome the inhibitory effects of dGTP and dTTP on CDP reduction, thereby circumventing a depletion of the dCTP pool. A partial reversal of deoxyadenosine toxicity was also obtained with addition of cytidine. In this case little change in the dCTP level was observed, but a decreased dGTP pool appeared to be correlated with growth inhibition. High cytidine ribonucleotide levels partially prevented this effect. The present results may encourage the use of cytidine in combination with deoxycytidine as a pharmacological regime in treatment of immunodeficiency disease associated with increased deoxyribonucleotide levels.

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“…An added complication is provided by the observation that "F-NMR chemical shifts are highly sensitive to changes in pH, temperature, solvent, and ionic strength (3, 4). We produced fluorinated uridine compounds by enzymatically converting (5,6) FUTP (produced by cells exposed to FUrd) in N-417 cell extracts to FUDP, FUDP-Glc, and FUDP-GlcA. Based on the associated changes in the 31P-and lgF-NMR spectra, we have definitively identified the chemical shifts of these compounds, as well as those of FUDP-GlcNAc and FUDPGalNAc.…”

Section: Introductionmentioning

confidence: 99%

Discrimination of fluorinated uridine metabolites in N‐417 small cell lung cancer cell extracts via ¹⁹F‐ and ³¹P‐NMR

Pederson

Zanghi

Miller

et al. 1994

Magnetic Resonance in Med

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Methods for the complete analysis of 5-fluorouracil metabolites in cell extracts

Cited by 50 publications

References 17 publications

p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

On the mechanism of deoxyribonucleoside toxicity in human T-lymphoblastoid cells. Reversal of growth inhibition by addition of cytidine

Discrimination of fluorinated uridine metabolites in N‐417 small cell lung cancer cell extracts via ¹⁹F‐ and ³¹P‐NMR

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Methods for the complete analysis of 5-fluorouracil metabolites in cell extracts

Cited by 50 publications

References 17 publications

p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

On the mechanism of deoxyribonucleoside toxicity in human T-lymphoblastoid cells. Reversal of growth inhibition by addition of cytidine

Discrimination of fluorinated uridine metabolites in N‐417 small cell lung cancer cell extracts via 19F‐ and 31P‐NMR

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Discrimination of fluorinated uridine metabolites in N‐417 small cell lung cancer cell extracts via ¹⁹F‐ and ³¹P‐NMR