Uptake and differential degradation of p53 mutants by dendritic cells from necrotic Saos-p53 positive cells.

S.E.B. McArdle, R. Germack, Selman Ali, R. C Rees.

Nottingham Trent University, Nottingham, Nottinghamshire United Kingdom

The tumour suppressor gene p53 is pivotal in the regulation of program cell death (apoptosis), and point mutations within the gene are the most common genetic alterations in human cancers. This process results in the overexpression of mutated and/or wild-type p53 protein. Cytotoxic T lymphocytes (CTL) play a pivotal role in the immune defense and mediate lysis (cytotoxicity) of target cells expressing appropriate MHC-peptide complexes. This response is however only possible if adequate T helper cell activation occurs. Specialised antigen presenting cells (APC) process protein and present antigenic peptide fragments on MHC class-II molecules to CD4+ T helper lymphocytes, which in turn provide T-cell help to generate CTL. Here we report, using both Western-Blotting and confocal microscopy, that the p53 protein from necrotic p53-transfected Saos-2 cells is taken up by immature dendritic cells (DC). Using Western-Blotting we demonstrated that this process was time and the activation status of the DC-dependant. No differences were found in the ability of DC to take up p53-positive-necrotic Saos-2 cells expressing two different protein conformational mutations (at position 175 or 273) but preliminary results indicate a difference in the kinetics of their degradation. Maturation of DC was induced by uptake of necrotic cells (Saos-2 lysate), which was increased by the addition of TNF alpha, and DC pulsed with lysate were able to induce autologous DR-restricted T cell proliferation. These results demonstrate for the first time that the uptake and degradation of p53 protein, a tumour associated antigen, by DC can elicit an immune response and that the conformation of the p53 protein may influence the kinetics of its degradation.

KEY WORDS: p53, dendritic cells.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Immunotherapy.