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 (NaAsO(2), CdSO(4).8H(2)O, Na(2)CrO(4).4H(2)O, MnCl(2), (NH(4))(2)PtCl(6)) 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;  ESKES Chantra;  STINGELE Silvia;  GARTLON Joanne;  FARINA Massimo;  PONTI Jessica;  PRICE Anna;  SABBIONI Enrico;  HARTUNG Thomas;  COECKE Sandra; 

2007-06-25

PERGAMON-ELSEVIER SCIENCE LTD

JRC37498

0887-2333,   

https://publications.jrc.ec.europa.eu/repository/handle/JRC37498,   

10.1016/j.tiv.2006.12.004,