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Updated: Oct 7, 2026
A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1
Published on: April 16, 2021
Interference of gemcitabine triphosphate with the measurements of deoxynucleotides using an optimized DNA polymerase
K Smid1, C J Van Moorsel, P Noordhuis
1Department of Medical Oncology, Vrije Universiteit Medical Centre (VUMC), PO Box 7057, 1007 MB Amsterdam, The Netherlands.
Abstract:
The main mechanism of action of the anticancer drug gemcitabine is assumed to be incorporation of its triphosphate (dFdCTP) into DNA, resulting in inhibition of DNA polymerization, inhibition of DNA synthesis and repair. Another mechanism is inhibition of ribonucleotide reductase leading to imbalance in the deoxyribonucleotide (dNTP) pools. One assay to measure dNTP pools is based on oligonucleotide elongation mediated by DNA polymerase. Since the latter may be affected by dFdCTP, we studied the effect of 0.1-600 pmol dFdCTP on this assay; 10 pmol and more dFdCTP significantly increased the average dpm of the blank (absence of other dNTP) and that of the calibration line of dATP (1.4-1.6-fold); 0.1 pmol and more increased that of the standard dGTP curve significantly (1.1-1.8-fold); 10-75 pmol decreased that of dCTP while 75 and 100 pmol significantly increased that of dCTP (1.3-fold); 50 pmol significantly increased that of dTTP (1.3-1.5-fold). For dATP, dGTP and dTTP, a saturation was reached at 100 pmol dFdCTP, but not yet for dCTP. To minimize these effects, we added an excess of 200 pmol dFdCTP to all samples and calibration lines when measuring dNTP levels of gemcitabine treated samples. In this way the effects of gemcitabine on dNTP levels were studied in human A2780 ovarian, HT29 colon, K562 myelogenous leukemia, H322 non-small cell lung cancer cell lines and the murine lung cancer cell line Lewis Lung. In all cell lines, intrinsic dTTP pools (3-77 pmol/106 cells) were the highest, followed by dATP (1.5-31), dCTP (0.7-27) and (nd-14) dGTP. Exposure to 1 and 10 microM gemcitabine for 4-h concentration dependently decreased dATP 3-10-fold and dGTP to undetectable levels, but dCTP at most 3-fold, while dTTP increased. In conclusion, dFdCTP affects dNTP measurements with the DNA polymerase elongation assay, but its effect could be controlled by addition of similar amounts of dFdCTP to each assay.
Insights
Gemcitabine
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Gemcitabine is an anticancer drug whose mechanism involves deoxyribonucleotide (dNTP) pool imbalance.
- Measuring dNTP pools is crucial for understanding gemcitabine's effects.
- DNA polymerase-based assays for dNTPs can be influenced by gemcitabine's active triphosphate form (dFdCTP).
Purpose of the Study:
- To investigate the impact of dFdCTP on a DNA polymerase elongation assay used for measuring dNTP pools.
- To establish a method for accurately measuring dNTP levels in gemcitabine-treated cancer cell lines.
Main Methods:
- Studied the effect of varying concentrations of dFdCTP (0.1-600 pmol) on DNA polymerase activity in vitro.
- Developed a modified assay by adding excess dFdCTP (200 pmol) to samples and calibration lines.
- Applied the optimized assay to measure dNTP levels in human ovarian, colon, leukemia, lung cancer cell lines, and a murine lung cancer cell line after gemcitabine exposure.
Main Results:
- dFdCTP significantly affected the assay's accuracy for all four dNTPs (dATP, dGTP, dCTP, dTTP) at different concentrations.
- A saturation effect was observed for dATP, dGTP, and dTTP with increasing dFdCTP, but not for dCTP.
- The addition of excess dFdCTP to the assay minimized these interfering effects.
- Gemcitabine treatment (1-10 microM) concentration-dependently decreased dATP and dGTP, minimally affected dCTP, and increased dTTP in various cancer cell lines.
Conclusions:
- dFdCTP interferes with DNA polymerase elongation assays for dNTP pool measurement.
- Adding excess dFdCTP to the assay is a viable strategy to control for these effects.
- This optimized method allows for reliable assessment of gemcitabine's impact on dNTP pools across different cancer cell types.
