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.