Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique

Zhang, Wei; Tan, Sheng-Lan; Paintsil, Elijah; Dutschman, Ginger E.; Gullen, Elizabeth A.; Chu, Edward; Cheng, Yung Chi

doi:10.1016/j.bcp.2011.05.009

Cited by 39 publications

(43 citation statements)

References 22 publications

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“…ATP and adenosine were measured from the CM directly after filtering through a_5,000 NMWL Ultrafree-MC filter unit (Millipore, Bedford, MA) containing adenosine-13C10, 15N5-triplosphate (ATP13C, 15N) as internal standard, modified from Zhang et al (2011) 34 . LC-MS analysis was conducted on a Thermo Fisher Q Exactive mass spectrometer with on-line separation using a Thermo Fisher/Dionex Ultimate 3000 HPLC.…”

Section: Methodsmentioning

confidence: 99%

Differential impact of adenosine nucleotides released by osteocytes on breast cancer growth and bone metastasis

et al. 2014

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Extracellular ATP has been shown to either inhibit or promote cancer growth and migration; however the mechanism underlying this discrepancy remained elusive. Here, we demonstrate the divergent roles of ATP and adenosine released by bone osteocytes in breast cancers. We showed that conditioned media (CM) collected from osteocytes treated with alendronate (AD), a bisphosphonate drug, inhibited the migration of human breast cancer MDA-MB-231 cells. Removal of the extracellular ATP by apyrase in CM abolished this effect, suggesting the involvement of ATP. ATP exerted its inhibitory effect through the activation of purinergic P2X receptor signaling in breast cancer cells evidenced by the attenuation of the inhibition by an antagonist, oxidized ATP, as well as knocking down P2X07 with siRNA, and the inhibition by an agonist, BzATP. Intriguingly, ATP had a biphasic effect on breast cancer cell behavior–lower dosage inhibited, but higher dosage promoted its migration. The stimulatory effect on migration was blocked by an adenosine receptor antagonist, MRS1754, ARL67156, an ecto-ATPase inhibitor, and A2A receptor siRNA, suggesting that in contrast to the action of ATP, adenosine, a metabolic product of ATP, promoted migration of breast cancer cells. Consistently, non-hydrolyzable ATP, ATPγS, only inhibited, but did not promote cancer cell migration. ATP also had a similar inhibitory effect on the Py8119 mouse mammary carcinoma cells; however, adenosine had no effect due to the absence of the A2A receptor. Consistent with the results of cancer cell migration, ATPγS inhibited, while adenosine promoted anchorage-independent growth of breast cancer cells. Our in vivo xenograft study showed a significant delay of tumor growth with the treatment of ATPγS. Moreover, the extent of bone metastasis in a mouse intratibial model was significantly reduced with the treatment of ATPγS. Together, our results suggest the distinct roles of ATP and adenosine released by osteocytes, and the activation of corresponding receptors P2X7 and A2A signaling on breast cancer cell growth, migration and bone metastasis.

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Section: Methodsmentioning

confidence: 99%

Differential impact of adenosine nucleotides released by osteocytes on breast cancer growth and bone metastasis

et al. 2014

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“…Although many methods relative to endogenous nucleotides separation and detection have been published [4,9,10], only a few described a complete analytical validation of the method [3,11]. Most of the articles presented calibration, accuracy, precision, but no results on stability of nucleotides was reported [8,12].…”

Section: Introductionmentioning

confidence: 94%

“…With the reequilibration steps, the total duration of a run is 37 min, representing a significant time saving compared to methods having chromatographic run times higher than 60 min [3,4,10,27].…”

Section: Mobile Phasementioning

confidence: 99%

“…Ion-pairing agent Several methods for the quantification of nucleotides used an ion-pairing agent in the mobile phase [3][4][5]. However, ion-pairing agents are known to reduce sensitivity and to be a cause of ion suppression [6,20].…”

Section: Optimization Of Liquid Chromatographymentioning

confidence: 99%

“…Therefore, many chromatographic methods based on anion exchange or ion-pairing (IP) mechanisms have been developed over the last years [2][3][4][5]. In IP chromatography, stationary phases are usually C18 or porous graphitic carbon (PGC).…”

Section: Introductionmentioning

confidence: 99%

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Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

et al. 2014

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An analytical method coupling online solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to quantify 16 endogenous nucleoside mono- and triphosphates in cellular samples. Separation was achieved on a porous graphitic carbon (PGC) column without ion-pairing agent in the mobile phase. Low levels of the ion-pairing agent diethylamine (DEA) added to the reconstitution solution were necessary to prevent peak tailing of nucleoside triphosphates. The mass spectrometer, a triple quadrupole with an electrospray ionisation source, was operated in positive mode. Two multiple reaction monitoring (MRM) segments were programmed, each an internal standard. Extraction and separation of nucleoside mono- and triphosphates were obtained within 20 min. The total duration of a single run was 37 min. Calibration curves, performed with labelled nucleotides added to the sample matrix, ranged from 0.29 to 18.8 pmol injected for deoxyribonucleotides and from 3.9 to 3,156 pmol for ribonucleotides. Accuracy did not deviate more than -14.6 and 10.2 % from nominal values for all compounds at all levels. CV results were all lower than 17.0 % for the LLOQ level and 14.6 % for the other levels. Quality control (QC) samples were also in agreement with acceptance criteria, except for the lower QC of GMP. Ion suppression, matrix effect, extraction recoveries and stability were assessed. After validation, the method was applied to the evaluation of the effects of gemcitabine and hydroxyurea on nucleotide pools in Messa cells.

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Nucleoside/Nucleotide Biomarkers

Zhang

2017

Targeted Biomarker Quantitation by LC–MS

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Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique

Cited by 39 publications

References 22 publications

Differential impact of adenosine nucleotides released by osteocytes on breast cancer growth and bone metastasis

Differential impact of adenosine nucleotides released by osteocytes on breast cancer growth and bone metastasis

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

Nucleoside/Nucleotide Biomarkers

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