IMP–GMP specific cytosolic 5′-nucleotidase regulates nucleotide pool and prodrug metabolism

• Published in: Biochimica et biophysica acta Vol. 1850; no. 7; pp. 1354 - 1361
• Main Authors: Cividini, Federico, Filoni, Daniela Nicole, Pesi, Rossana, Allegrini, Simone, Camici, Marcella, Tozzi, Maria Grazia
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2015
• Subjects: 5'-Nucleotidase - genetics; 5'-Nucleotidase - metabolism; antineoplastic agents; Antineoplastic Agents - metabolism; Antineoplastic Agents - pharmacology; capillary electrophoresis; Cell Survival - drug effects; Cell Survival - genetics; Chemotherapy; cN-II; Cytarabine; Cytarabine - metabolism; Cytarabine - pharmacology; cytotoxicity; Deoxycytidine - analogs & derivatives; Deoxycytidine - metabolism; Deoxycytidine - pharmacology; dephosphorylation; Dose-Response Relationship, Drug; drug resistance; Drug Resistance - genetics; enzyme activity; Fludarabine; Gemcitabine; green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Guanosine Monophosphate - metabolism; HEK293 Cells; Humans; hydrolysis; Immunoblotting; inhibitory concentration 50; Inosine Monophosphate - metabolism; nucleosides; Nucleotides - metabolism; patients; pharmacokinetics; Phosphorylation - drug effects; Prodrugs - metabolism; Prodrugs - pharmacology; prognosis; protein-protein interactions; Resistance; resistance mechanisms; Substrate Specificity; vector control; Vidarabine - analogs & derivatives; Vidarabine - metabolism; Vidarabine - pharmacology; CDA; AMP-PCP; Gemcitabine; PRPP; dNTP; HGPRT; MTT; dCK; Ipaf; Resistance; DMEM; Chemotherapy; PNP; TCA; Fludarabine; Cytarabine; cN-II; BPG; GAPDH; ADA
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2015.03.017

Type II cytosolic 5′-nucleotidase (cN-II) catalyzes the hydrolysis of purine and, to some extent, of pyrimidine monophosphates. Recently, a number of papers demonstrated the involvement of cN-II in the mechanisms of resistance to antitumor drugs such as cytarabine, gemcitabine and fludarabine. Furthermore, cN-II is involved in drug resistance in patients affected by hematological malignancies influencing the clinical outcome. Although the implication of cN-II expression and/or activity appears to be correlated with drug resistance and poor prognosis, the molecular mechanism by which cN-II mediates drug resistance is still unknown. HEK 293 cells carrying an expression vector coding for cN-II linked to green fluorescent protein (GFP) and a control vector without cN-II were utilized. A highly sensitive capillary electrophoresis method was applied for nucleotide pool determination and cytotoxicity exerted by drugs was determined with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Over-expression of cN-II causes a drop of nucleoside triphosphate concentration and a general disturbance of nucleotide pool. Over-expressing cells were resistant to fludarabine, gemcitabine and cytarabine independently of cN-II ability to hydrolyze their monophosphates. An increase of cN-II expression is sufficient to cause both a general disturbance of nucleotide pool and an increase of half maximal inhibitory concentration (IC50) of the drugs. Since the monophosphates of cytarabine and gemcitabine are not substrates of cN-II, the protection observed cannot be directly ascribed to drug inactivation. Our results indicate that cN-II exerts a relevant role in nucleotide and drug metabolism through not only enzyme activity but also a mechanism involving a protein–protein interaction, thus playing a general regulatory role in cell survival. Resistance to fludarabine, gemcitabine and cytarabine can be determined by an increase of cN-II both through dephosphorylation of active drugs and perturbation of nucleotide pool. •Effect of cN-II over-expression on intracellular nucleotide content in a Human Embryonic Kidney 293 cell model•Cytotoxicity of fludarabine, gemcitabine and cytarabine as a function of cN-II over-expression.•Discussion on mechanisms of cell resistance to nucleoside analog antitumoral drugs