Development of a mechanistically-based genetically engineered PC12 cell system to detect p53-mediated cytotoxicity.
The human wild type p53 gene, key for apoptosis, was introduced into the pheochromocytoma (PC12) cell line, to create a mechanistically-based in vitro test model for the detection of p53-mediated toxicity. Expression of the wt p53 gene was regulated by a system, which allowed or blocked expression p53 by absence or presence of tetracycline in the culture media. Western blot analyses confirmed an inducible and tetracycline-dependent expression of the wt p53 protein. Functionality of the p53 protein was verified by camptothecin treatment, known to induce p53-dependent apoptosis. Results showed that p53-expressing cells were significantly more sensitive to camptothecin induced cytotoxicity compared to non-expressing cells, and presented a significantly higher incidence of apoptosis. A screening study on 31 metal compounds, showed that the classified human carcinogens (NaAsO2, CdSO4 .8H2O, Na2CrO4 .4H2O, MnCl2, (NH4)2PtCl6) significantly increased cytotoxicity in p53-expressing cells compared to non-expressing cells, suggesting that their cytotoxicity was p53-mediated. Finally, acute and subchronic treatment with methyl mercury showed no significant differences in cytotoxicity and the percentage of apoptosis or necrosis between p53-expressing and non-expressing differentiated cells, suggesting that methyl mercury cytotoxicity was p53-independent.
van Vliet
Erwin
E
European Centre for the Validation of Alternative Methods (ECVAM), Institute for Health and Consumer Protection, European Commission Joint Research Centre, Via E. Fermi 1, 21020 Ispra (VA), Italy. erwin.van-vliet@jrc.it
Eskes
Chantra
C
Stingele
Silvia
S
Gartlon
Joanne
J
Price
Anna
A
Farina
Massimo
M
Ponti
Jessica
J
Hartung
Thomas
T
Sabbioni
Enrico
E
Coecke
Sandra
S
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D018053:Arsenites / Q000633:toxicity; D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology; D002454:Cell Differentiation; D002470:Cell Survival / Q000187:drug effects* / Q000235:genetics*; D004264:DNA Topoisomerases, Type I / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D016158:Genes, p53 / Q000235:genetics*; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D007202:Indicators and Reagents; D008767:Methylmercury Compounds / Q000633:toxicity; D009498:Neurotoxins / Q000633:toxicity; D016716:PC12 Cells / Q000187:drug effects*; D051381:Rats; D059004:Topoisomerase I Inhibitors; D014131:Trace Elements / Q000494:pharmacology; D014162:Transfection
D000972:Antineoplastic Agents, Phytogenic; D018053:Arsenites; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008767:Methylmercury Compounds; D009498:Neurotoxins; D059004:Topoisomerase I Inhibitors; D014131:Trace Elements; D004264:DNA Topoisomerases, Type I; C015001:arsenite; D002166:Camptothecin