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

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.

Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents

“…A green fluorescent protein (GFP) gene expression vector driven by a cytomegalovirus promoter, peGFP-C1 (BD Biosciences, Clontech, CA, USA), carrying the neomycin resistance gene allowing the isolation of drug-resistant clones, was used as a marker for tracking gene transfection and expression. Expression vectors coding for shRNA against cN-II (pScN-II) and control sequence (pScont) were generously provided by Dr. Jordheim [ 30 ]. Plasmid DNA (pDNA) was amplified and purified using QIAprep Midiprep Kit (QIAGEN, CA, USA) according to the manufacturer’s protocol and dissolved in TE buffer (10 mM Tris-HCl, 1 mM ethylene-diaminetetra-acetic acid) at a concentration of 1 mg/ml.…”

Cell proliferation and drug sensitivity of human glioblastoma cells are altered by the stable modulation of cytosolic 5′-nucleotidase II

“…Overexpression induced in both cases a decrease of nucleotide triphosphates and an increase of duplication time . When stably inhibited in human cancer cells from hematologic origin, no difference in proliferation or distribution in cell cycle was observed (Jordheim et al, 2015, in press), whereas these cells were more sensitive to nucleoside analogs such as fludarabine (Supplemental Fig. 1).…”

The cytosolic 5′-nucleotidase cN-II lowers the adaptability to glucose deprivation in human breast cancer cells

“…The cytosolic enzyme cN-II has a preference for IMP and GMP and has also been described as being capable of phosphorylating nucleosides through a phosphotransferase activity [ 7 ]. We have previously shown that this enzyme is involved in the sensitivity of cancer cells to nucleoside analogue-based chemotherapy [ 8 , 9 ], and developed and studied enzymatic inhibitors [ 10 – 14 ]. The clinical relevance of this approach has been confirmed by the observation of hyperactive cN-II mutants in relapsed pediatric acute lymphoblastic leukemia patients associated with a resistance to purine analogues [ 15 , 16 ].…”