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NMP, NDP, NTP, dNTP Quantification by HPLC

Ref. # WCS-Nov 6
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Download "HPLC Analysis RNR Inhibitors" 

Cellular Assay for RNR Inhibitors

This assay has been specially tailored to validate Ribonucleotide Reductase (RNR) Inhibition by a given compound in cultured cells. After incubation of cultured cells with the inhibitor, this high performance liquid chromatography analysis of nucleotides consists in:
  • Extraction of NMP, NDP, NTP, and dNTP by SPE (Solid Phase Extraction) procedure
  • Separation by ion-paired HPLC of 18-22 nucleotides (Agilent 1100)
  • Quantification by ion-paired UV-HPLC (peak area at 254nm)

This cellular assay for Ribonucleotide Reductase (RNR) Inhibition was validated with hydroxyurea and gemcitabine in HeLa cultured cells. .

Example 1: Hydroxyurea (HU)
Hydroxyurea is an antineoplastic agent, antimetabolite, used to treat melanoma, chronic myelocytic leukemia and certain blood disorders. Hydroxyurea is known to inhibit DNA synthesis by destroying the catalytically essential free radical of class I ribonucleoside diphosphate (rNDP) reductase, thereby blocking the de novo synthesis of deoxyribonucleotides. In mammalian cells, hydroxyurea treatment causes a differential depletion of the four deoxyribonucleoside triphosphate pools with dATP being most severely depleted(1, 2). As illustrated by Figure 1, hydroxyurea treatment induces in HeLa cells profound depletion of deoxyadenosine triphosphate and significant loss of dADP, dUDP and dTTP, which is consistent with previously published data(1, 2).

Figure 1. Nucleotide profiles of hydroxyurea-treated HeLa cells (1mM, 20h)
Ratio between nucleotide content in drug-treated and untreated cells are shown

Figure 2. Effect of hydroxyurea on cellular pool of deoxynucleotides.
The depleted nucleotides are shown in red. Ribonucleotide reductase (RNR), a recognized target of hydroxyurea, is framed in red.

Figure 3. Superposition of HPLC spectra of nucleotides extracted from HeLa cells treated with 1mM hydroxyurea (red) and DMSO (blue).
Focus on depletion in dUDP and dADP is shown on left and in dTTP and dATP on right.

Example 2: Gemcitabine (dFdC)
Gemcitabine (2’,2’-difluorodeoxycytidine, dFdC) is a nucleoside analogue clinically used as an anticancer prodrug. Its phosphorylated metabolites target numerous cellular enzymes involved in nucleotide biosynthesis, including ribonucleotide reductase (RNR) which is strongly inhibited by diphosphorylated form of gemcitabine, dFdCDP. As shown in Figure 4, major changes in nucleotides induced by dFdC in HeLa cells concern the depletion in cellular dATP, dTTP, dGTP and dUDP due to RNR inhibition. The depletion of cellular dUMP indicates inhibition of dCMP-deaminase consistently with previously reported data(3), but may also reflect the decrease in cellular dUDP, a source of dUMP.

Figure 4. Nucleotide profiles of gemcitabine-treated HeLa cells (37µM, 20h)
Ratio between nucleotide content in drug-treated and untreated cells are shown

Figure 5. Effect of gemcitabine on cellular pool of nucleotides and deoxynucleotides.
The depleted (<50% of control) nucleotides are shown in red. Ribonucleotide reductase (RNR) and dCMP-deaminase (dCMP-DA), recognized targets of gemcitabine, are framed in red.

Figure 6. Superposition of HPLC spectra of nucleotides extracted from HeLa cells treated with 37µM gemcitabine (blue) and DMSO (red) illustrating depletion in dNTP.

1. V. Bianchi et al. (1986): Changes of Deoxyribonucleoside Triphosphate Pools Induced by Hydroxyurea and Their Relation to DNA Synthesis J. Biol Chem. 261(34), 16037-42
2. S. P. Hendricks and C. K. Mathews (1998): Differential Effects of Hydroxyurea upon Deoxyribonucleoside Triphosphate Pools, Analyzed with Vaccinia Virus Ribonucleotide Reductase J. Biol Chem. 273(45), 29519-23
3. C. J. van Moorsel et al. (2000): Differential effects of gemcitabine on ribonucleotide pools of twenty-one solid tumour and leukaemia cell lines Biochem. Biophys. Acta. 1474(1), 5-12
et al.(1995): An Ion-Pairing High-Performance Liquid Chromatographic Method for the Direct Simultaneous Determination of Nucleotides, Deoxynucleotides, Nicotinic Coenzymes, Oxypurines, Nucleosides, and Bases in Perchloric Acid Cell Extracts Anal. Biochem. 231(2), 407-412

Download this document "Cell-based RNR inhibiton assay" 

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