Acylphosphatase overexpression triggers SH-SY5Y differentiation towards neuronal phenotype.
An acylphosphatase (AcPase) overexpression study was carried out on SH-SY5Y neuroblastoma cells, using a green fluorescent fusion protein (AcP-GFP), with GFP acting as a reporter protein. The cellular proliferation rate was significantly reduced by overexpression of AcPase by a factor of ten. In contrast, clones transfected with two inactive AcPase mutants showed a growth rate comparable to control cells. This suggests that AcPase catalyzes the proliferative down-regulation. AcPase-overexpressing clones showed a physiological mortality rate as assessed by an MTT reduction test and by evaluation of necrotic markers. DNA fragmentation analysis and assays of caspase-3 and poly (ADP-ribose) polymerase (PARP)-active fragments showed no evidence of any apoptotic pattern. AcPase overexpression led to a marked increase in PARP activity as well as Bcl-2 content; these are commonly up-regulated during differentiative processes in neuronal cells. In fact, the typical differentiation marker, growth-associated-protein 43, was significantly up-regulated. Microscopic observations also showed a clear increase in the differentiative phenotype in AcPase-overexpressing cells. Our results clearly show that AcPase plays a primary causative role in neuronal differentiation.
Cecchi
C
C
Department of Biochemical Sciences, University of Florence, Viale Morgagni 50, 50134, Italy.
Liguri
G
G
Fiorillo
C
C
Bogani
F
F
Gambassi
M
M
Giannoni
E
E
Cirri
P
P
Baglioni
S
S
Ramponi
G
G
D017766:Acid Anhydride Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D017209:Apoptosis / Q000502:physiology; D015415:Biomarkers; D002454:Cell Differentiation / Q000502:physiology*; D002455:Cell Division; D017930:Genes, Reporter; D009447:Neuroblastoma / Q000378:metabolism*; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D015415:Biomarkers; D011993:Recombinant Fusion Proteins; D000097024:Acylphosphatase