•
RNR Inhibition - Whole Cell Screening
To study the effect of nucleoside analogues on the whole spectra of cellular purine and pyrimidine ribo- and deoxyribonucleotides, NOVOCIB has developed an original cell-based analytical approach and a range of whole cell assays in which more than 31 (deoxy)ribonucleotides (mono-, di-, triphosphate) and nucleotide co-factors are extracted from cultured cells, separated by ion-pared chromatography and quantified.
Our Whole Cell Screening for Ribonucleotide Reductase (RNR) Inhibition was validated with hydroxyurea and gemcitabine.
To study the effect of nucleoside analogues on the whole spectra of cellular purine and pyrimidine ribo- and deoxyribonucleotides, NOVOCIB has developed an original cell-based analytical approach and a range of whole cell assays in which more than 31 (deoxy)ribonucleotides (mono-, di-, triphosphate) and nucleotide co-factors are extracted from cultured cells, separated by ion-pared chromatography and quantified.
Our Whole Cell Screening for Ribonucleotide Reductase (RNR) Inhibition was validated with hydroxyurea and gemcitabine.
Aim: This Research Contract Service has been specially tailored to validate Inosine Monophosphate Dehydrogenase (IMPDH) inhibition by a given compound in cultured cells. It consists in extracting, identifying and quantifying by HPLC the intracellular concentration of deoxynucleotides di- and triphosphate in compound-treated cells
Whole Cell Screening for RNR Inhibition was validated with hydroxyurea and gemcitabine in HeLa cultured cells.
IMPORTANT: Client-specified alterations can be accommodated
1st Example: 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 V mouse over to enlarge
|
 |
 |
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. |
|
mouse over to enlarge V
|
V mouse over to enlarge
|
 |
 |
2nd Example: 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 V mouse over to enlarge
|
 |
|
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.
V mouse over to enlarge
|
 |
Materials & Methods
Cells treatment: Huh-7 cells are grown in an atmosphere of humidified 5% CO2 at 37°C in DMEM medium supplemented with 2mM L-glutamine, 10% heat-inactivated fetal bovine serum and streptromycin-penicillin. Exponentially grown Huh-7 cells are seeded at ≈6x105 cells per 10cm cell-culture dish. After 48h of growth, the culture medium is replaced with fresh FCS-supplemented medium followed by addition of 10µL of DMSO or DMSO-dissolved compound.
Extraction of nucleotides and deoxynucleotides - Sample preparation: The nucleotides are extracted from cell monolayers by addition of 3 ml per dish of ice-clod 80% acetonitrile for 1h. The extracts are centrifuged to remove cellular debris and nucleotides are extracted by SPE procedure (SAX column, Supelco, Sigma-Aldrich) pre-conditioned with methanol, water and acetonitrile. The eluent is filtered through 0.45µm filter membrane (Roth) and analyzed by HPLC
Analytical system: 1) An Agilent 1100 series liquid chromatograph fitted with binary pump G1312A, vacuum degasser G1322A, well-plate autosampler G1367A, thermostated column compartment G1316A and multiple wavelenght and diode array detector G1315B. Run and data acquisition are controlled by Agilent ChemStation software.
2) Zorbax Extend-C18 4.6x150mm, 3.5µm particle size and corresponding guard column (Agilent). 5µl of cell extract were analyzed using Zorbax Extend-C18 column by ion-pairing HPLC method reported previously for the simultaneous separation and quantification of bases, nucleosides and nucleotides(4) with slight modifications as follows.
HPLC calibration, peak identification and quantification: Calibrations are performed with standards prepared in mobile phase and with standards mixed with cell extracts, which are run immediately before and after every series of samples. Assignment of the peaks that correspond to different deoxyribonucleosides and ribonucleosides mono-, di-, and triphosphate of the cell extract spectrum is done by comparing both retention times and characteristics of UV absorption spectra (254/280 ratio) with those of standards. The area of individual peaks was measured using ChemStation software (Agilent).
Download this document "RNR inhibition: Whole Cell Assay" 
Ask for Quotation