p53 status of newly established acute myeloid leukaemia cell lines

We analysed the status of the p53 gene and protein in eight newly established acute myeloid leukaemia (AML) cell lines representing blast cells of either de novo leukaemia patients in first remission or patients with relapsed and chemotherapy-resistant disease causing their death. There were no mutations in the p53 gene in any of the cell lines as analysed by single-strand conformation polymorphism of amplified exons 5–8. However, the p53 protein was clearly and consistently expressed in all of these cell lines, as shown by immunohistochemistry, Western blotting and flow cytometry. The consistently expressed p53 protein was located in both the nucleus and the cytoplasm of all the cell lines and, as shown by flow cytometry, it was mostly in a conformation typical of the mutated protein. These AML cell lines offer a tool for studying the production and function of the p53 protein and its possible role in cell cycle regulation and chemoresistance as well as in the regulation of apoptosis in AML. © 1999 Cancer Research Campaig

Conformational Altered p53 as an Early Marker of Oxidative Stress in Alzheimer's Disease

In order to study oxidative stress in peripheral cells of Alzheimer's disease (AD) patients, immortalized lymphocytes derived from two peculiar cohorts of patients, referring to early onset AD (EOSAD) and subjects harboured AD related mutation (ADmut), were used. Oxidative stress was evaluated measuring i) the typical oxidative markers, such as HNE Michel adducts, 3 Nitro-Tyrosine residues and protein carbonyl on protein extracts, ii) and the antioxidant capacity, following the enzymatic kinetic of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRD). We found that the signs of oxidative stress, measured as oxidative marker levels, were evident only in ADmut but not in EOSAD patients. However, oxidative imbalance in EOSAD as well as ADmut lymphocytes was underlined by a reduced SOD activity and GRD activity in both pathological groups in comparison with cells derived from healthy subjects. Furthermore, a redox modulated p53 protein was found conformational altered in both EOSAD and ADmut B lymphocytes in comparison with control cells. This conformational altered p53 isoform, named “unfolded p53”, was recognized by the use of two specific conformational anti-p53 antibodies. Immunoprecipitation experiments, performed with the monoclonal antibodies PAb1620 (that recognizes p53wt) and PAb240 (that is direct towards unfolded p53), and followed by the immunoblotting with anti-4-hydroxynonenal (HNE) and anti- 3-nitrotyrosine (3NT) antibodies, showed a preferential increase of nitrated tyrosine residues in unfolded p53 isoform comparing to p53 wt protein, in both ADmut and EOSAD. In addition, a correlation between unfolded p53 and SOD activity was further found. Thus this study suggests that ROS/RNS contributed to change of p53 tertiary structure and that unfolded p53 can be considered as an early marker of oxidative imbalance in these patients