Knockdown of cytosolic 5'-nucleotidase II (cN-II) reveals that its activity is essential for survival in astrocytoma cells.
IMP preferring cytosolic 5'-nucleotidase (cN-II) is an ubiquitous nucleotide hydrolysing enzyme. The enzyme is widely distributed and its amino acid sequence is highly conserved among vertebrates. Fluctuations of cN-II activity have been associated with the pathogenesis of neurological disorders. The enzyme appears to be involved in the regulation of the intracellular availability of the purine precursor IMP and also of GMP and AMP, but the contribution of this activity and of its regulation to cell metabolism and to CNS cell functions remains uncertain. To address this issue, we used a vector based short hairpin RNA (shRNA) strategy to knockdown cN-II activity in human astrocytoma cells. Our results demonstrated that 53 h after transduction, cN-II mRNA was reduced to 17.9+/-0.03% of control cells. 19 h later enzyme activity was decreased from 0.7+/-0.026 mU/mg in control ADF cells to 0.45+/-0.046 mU/mg, while cell viability (evaluated by the MTT reduction assay) decreased up to 0.59+/-0.01 (fold vs control) and caspase 3 activity increased from 136+/-5.8 pmol min(-1) mg(-1) in control cells to 639+/-37.5 pmol min(-1) mg(-1) in silenced cells, thus demonstrating that cN-II is essential for cell survival. The decrease of enzyme activity causes apoptosis of the cultured cells without altering intracellular nucleotide and nucleoside concentration or energy charge. Since cN-II is highly expressed in tumour cells, our finding offers a new possible therapeutical approach especially against primary brain tumours such as glioblastoma, and to ameliorate chemotherapy against leukemia.
Careddu
Maria Giovanna
MG
Dipartimento di Biologia, Università di Pisa, Via S. Zeno 51, 56127 Pisa, Italy.
Allegrini
Simone
S
Pesi
Rossana
R
Camici
Marcella
M
Garcia-Gil
Mercedes
M
Tozzi
Maria Grazia
MG
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D001254:Astrocytoma / Q000201:enzymology* / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D006801:Humans; D011687:Purines / Q000378:metabolism; D034622:RNA Interference; D051381:Rats
D011687:Purines; D015720:5'-Nucleotidase; C508048:NT5C2 protein, human