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S Ulisse, Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy
Y Arlot-bonnemains, Genetiqué et developpement, CNRS, Rennes, France
E Baldini, Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy
S Morrone, Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy
S Carocci, Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy
L Di Luigi, Department of Health Sciences, "Foro Italico" University of Rome, Rome, Italy
M D'Armiento, Department of Experimental Medicine, "Sapienza" University of Rome , Rome, 00161, Italy

Correspondence: Massimino D'Armiento, Email: massimino.darmiento{at}uniroma1.it

The Aurora kinases family members, Aurora-A, -B and –C, are serine/threonine kinases involved in the regulation of chromosome segregation and cytokinesis, and alterations in their expression are associated with malignant cell transformation and genomic instability. Deregulation of the expression of the Aurora kinases has been shown to occur also in testicular germ cell tumors (TGCT) identifying them as putative anti-cancer therapeutic targets. We here evaluated the in vitro effects of MK-0457, an Aurora kinases inhibitor, on cell proliferation, cell cycle, ploidy, apoptosis and tumorigenicity on the TGCT-derived cell line NT2-D1. Treatment with MK-0457 inhibited cell proliferation in a time- and dose-dependent manner, with IC50 = 17.2±3.3 nM. MK-0457 did not affect the expression of the three Aurora kinases, but prevented their ability to phosphorylate substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it, presenting after short time the typical features of apoptotic cells. Cytofluorimetric analysis confirmed that the treatment with MK-0457 for 6h induced NT2-D1 cells accumulation in the G2/M phase of the cell cycle and the subsequent appearance of sub-G0 nuclei. The latter result was further supported by the detection of caspase-3 activation following 24 h treatment with the inhibitor. Finally, MK-0457 prevented the capability of the NT2-D1 cells to form colonies in soft agar. In conclusion, the above findings demonstrate that inhibition of Aurora kinases activity is effective in reducing in vitro growth and tumorigenicity of NT2-D1 cells, and indicate its potential therapeutic value for TGCT treatment.