Involvement of Hsp70, a stress protein, in the resistance of long-term culture of PC12 cells against sodium nitroprusside (SNP)-induced cell death.
Sodium nitroprusside (SNP)-treated PC12 cell line is being used in our laboratory as a cell model of nitric oxide (NO)-mediated damage for in vitro evaluation of potential neuroprotective compounds, thus cell response to SNP must be standardized to gain reproducible data. The NO-donor SNP has been shown to induce cell death at high concentrations in undifferentiated PC12 cells. Differences were found in sensitivity to SNP between cells from short- and long-term cultured cells. After 24-h exposure to 100-500 microM SNP, a decrease of cell viability was observed in both short- (17, 21 and 23rd passages) and long-term cultures (46, 49 and 50th passages), with IC(50) values of 312.72 and 462.90 microM, respectively. In cells from early passages, SNP-induced cell death was accompanied by significant increases of LDH leakage, nitrite production, malondialdehyde (MDA) levels, catalase (CAT) activity, cleavage of poly(ADP-ribose)polymerase (PARP) and caspase-3 activation in comparison with those from late passages. Furthermore, untreated and SNP-treated cells from long-term cultures displayed an increase of the stress protein Hsp70 levels when compared with those from short-term cultures. Up-regulated levels of Hsp70 may be associated with cell survival. Therefore, cells may acquire a certain resistance to SNP-induced toxicity associated with an increase in cell passage-dependent Hsp70. The protein Hsp70 might modulate the cellular response to the toxic insult by increasing CAT and GSH-Px activities and decreasing caspase-3 activation. Finally, it is crucial for the standardization of this cell model of neurotoxicity, at least in part, the use of PC12 cells in an optimum and reliable range of passages.
Romero
Carmen
C
Pharmacology Department, School of Pharmacy, Complutense University of Madrid, 28040 Madrid, Spain.
Benedí
Juana
J
Villar
Angel
A
Martín-Aragón
Sagrario
S
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D018929:Cell Culture Techniques; D016923:Cell Death; D002470:Cell Survival; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism* / Q000494:pharmacology; D018696:Neuroprotective Agents / Q000633:toxicity*; D009569:Nitric Oxide / Q000378:metabolism / Q000633:toxicity*; D020030:Nitric Oxide Donors / Q000633:toxicity; D009599:Nitroprusside / Q000633:toxicity*; D016716:PC12 Cells; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051381:Rats
D018840:HSP70 Heat-Shock Proteins; D018696:Neuroprotective Agents; D020030:Nitric Oxide Donors; D009599:Nitroprusside; D009569:Nitric Oxide; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3