Effect of the prion protein fragment hPrP[173-195] on the proliferation of B104 neuroblastoma cells.

Prion diseases are a group of neurodegenerative pathologies that recognize, as aetiopathologic agent, an aberrant isoform of the prion protein (PrPc), named PrPsc [1]. Recently, a correlation between the structural state of the prion protein and its neurotoxicity has been demonstrated and, by using different prion peptide fragments of the structured portion of the protein, the molecular mechanisms involved in the pathogenesis of prion diseases begin to be elucidated [2]. In this study, we examined the neurotoxicity of the prion protein fragment PrP[173-195] (Ac-NNFVHDCVNITIKQHTV-TTTTKG-NH2) on a neuronal cell line (B104 neuroblastoma cells). PrP[173-195] corresponds to a highly conserved region that could provide a nucleation site for the sequence-dependent unfolding and may be implicated in the conformational transition from PrPC to PrPSc. Stock cultures of B104 cells were maintained in Eagle's minimal essential medium (MEM) as described elsewhere [3]. B104 cells were plated into 96-well trays and maintained in the medium containing 10% fetal bovine serum (FBS) for at least 24 h, then, a cell aliquot was switched to a medium containing 0.1% FBS. Then, all cells were treated with increasing concentrations of PrP[173-195] up to 12 µg/ml and cell viability was assessed by MTT (3,[4,5dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) test after 18, 24 and 48 h of incubation with the peptide fragment. After 18 h incubation, a maximum of 30% reduction of cell viability was observed in all cells at peptide concentrations of 2 µg/ml. After 24 h incubation, the same concentration of peptide induced a maximum of reduction of almost 55% cell viability in 0.1% FBS, while in cell maintained in 10% FBS the toxic effect was maintained at the same level observed after 18 h incubation. After 48 h incubation, a continuous, concentration-dependent reduction of cell viability up to 90% (10% FBS) and 80% (0.1% FBS) was measured up to 12 µg/ml. To assess the specificity of the neurotoxic effects of the fragment, was assayed by performing the same analyses on human MCF7 breast cancer cell line.