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Determination of the enzymatic activity of cytosolic 5′-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models

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Abstract

The cytosolic 5′-nucleotidase (cN-II) has been shown to be involved in the response of cancer cells to cytotoxic agents, and the quantification of its activity in biological samples is of great interest. In this context, we developed and validated an analytical method for determination of cN-II activity in cultured cancer cells. This non-radioactive method, using a Hypercarb column as stationary phase, was validated with a lower limit of quantification of 0.1 μM inosine. We used it to characterize cell line models with modified cN-II expression obtained with stable transfections. We show that the short hairpin RNA (shRNA)-mediated inhibition of cN-II expression in various malignant blood cells is associated with decreased protein expression and enzymatic activity (1.7–6.2-fold) as well as an increased sensitivity to cytotoxic agents (up to 14-fold). On the other hand, expression of green fluorescent protein (GFP)-fused wild type or hyperactive mutant (R367Q) cN-II increased the activity and also decreased the sensitivity to nucleoside analogues. Our results confirm the biological relevance of modulating cN-II in cancer cells, and we present a straightforward validated method for the determination of cN-II activity in cellular samples.

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Abbreviations

ACN:

Acetonitrile

ALL:

Acute lymphoblastic leukemia

AML:

Acute myeloid leukemia

araA:

Arabinofuranosyladenine

BSA:

Bovine serum albumin

CLL:

Chronic lymphocytic leukemia

cN-II:

Cytosolic 5′-nucleotidase II

DTT:

Dithiothreitol

ESI:

Electrospray ionization

IS:

Internal standard

LC-MS/MS:

Liquid chromatography coupled to tandem mass spectrometry

LLOQ:

Lower limit of quantification

MTT:

Dimethylthiazolyl-diphenyltetrazolium bromide

PBS:

Phosphate buffer saline

QC:

Quality control

shRNA:

Short hairpin RNA

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Acknowledgments

This study was funded by the ANR “cN-II Focus” and the Région Languedoc-Roussillon “Chercheur d’Avenir 2009.” LPJ acknowledges Olav Raagholt og Gerd Meidel Raagholts stiftelse for forskning and Astri og Birger Torsteds legat. The authors are grateful to Eve Mattei for her technical assistance in the preparation of the patient samples and to Federico Cividini for PCR experiments.

Conflict of interest

The authors have no conflicts of interest.

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Authors and Affiliations

  1. Université de Lyon, 69000, Lyon, France

    Lars Petter Jordheim, Emeline Cros-Perrial & Charles Dumontet

  2. Université de Lyon 1, 69000, Lyon, France

    Lars Petter Jordheim, Emeline Cros-Perrial & Charles Dumontet

  3. INSERM U1052, Centre de Recherche en Cancérologie de Lyon, 69000, Lyon, France

    Lars Petter Jordheim, Emeline Cros-Perrial & Charles Dumontet

  4. CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon, 69008, Lyon, France

    Lars Petter Jordheim, Emeline Cros-Perrial & Charles Dumontet

  5. Centre Léon Bérard, 69008, Lyon, France

    Lars Petter Jordheim, Emeline Cros-Perrial & Charles Dumontet

  6. Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS - UM1 - UM2, Université Montpellier 2, Place Eugène Bataillon, 34095, Montpellier Cedex 5, France

    Jean-Yves Puy, Suzanne Peyrottes, Isabelle Lefebvre & Christian Périgaud

  7. Hospices Civils de Lyon, 69000, Lyon, France

    Charles Dumontet

Authors
  1. Lars Petter Jordheim
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  2. Jean-Yves Puy
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  3. Emeline Cros-Perrial
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  4. Suzanne Peyrottes
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  5. Isabelle Lefebvre
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  6. Christian Périgaud
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  7. Charles Dumontet
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Correspondence to Lars Petter Jordheim.

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Jordheim, L.P., Puy, JY., Cros-Perrial, E. et al. Determination of the enzymatic activity of cytosolic 5′-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models. Anal Bioanal Chem 407, 5747–5758 (2015). https://doi.org/10.1007/s00216-015-8757-4

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  • DOI: https://doi.org/10.1007/s00216-015-8757-4

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