Purpose: To simultaneously quantify intracellular nucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) pools and to assess their changes produced by interfering with ribonucleotide reductase (RNR) expression in leukemia cells. Methods: A HPLC-MS/MS system was used to quantify intracellular NTP and dNTP pools. Results: The assay was linear between 50 nM, the lower limit of quantification (LLOQ), and 10 μM in cell lysate. The within-day coefficients of variation (CVs, n∈=∈5) were found to be 12.0-18.0% at the LLOQ and 3.0-9.0% between 500 and 5,000 nM for dNTPs and 8.0-15.0% and 2.0-6.0% for NTPs. The between-day CVs (n∈=∈5) were 9.0-13.0% and 3.0-11.0% for dNTPs and 9.0-13.0% and 3.0-6.0% for NTPs. The within-day accuracy values were 93.0-119.0% for both NTPs and dNTPs. ATP overlapped with dGTP and they were analyzed as a composite. This method was applied to measure basal intracellular dNTPs/NTPs in five leukemia cell lines exposed to the RNR antisense GTI-2040. Following drug treatment, dCTP and dATP levels were found to decrease significantly in MV4-11 and K562 cells. Additionally, perturbation of dNTP/NTP levels in bone marrow sample of a patient treated with GTI-2040 was detected. Conclusions: This method provides a practical tool to measure intracellular dNTP/NTP levels in cells and clinical samples.
Bibliographical noteFunding Information:
This work was supported in part by the National Cancer Research Institute, Bethesda, MD, RO1 CA102031 (P.I. GM) and by the Biomedical Mass Spectrometry Laboratory, The Ohio State University.
- DNTP/NTP levels