Absence of p53 autoantibodies in a significant proportion of breast cancer patients.

We analysed antibodies specific for human p53 in sera from primary breast cancer patients using three different immunoassays and we related these results to the p53 level in tumour tissue detected by immunohistochemistry. Only 44% (11/25) of apparently enzyme-linked immunosorbent assay (ELISA)-positive sera were from patients with a high level of p53 protein in more than 50% of their tumour cells. Surprisingly, 36% (9/25) of the sera originated from patients with no detectable p53 protein at all. Immunoprecipitation data suggested that the reason for this discrepancy is that at least some of the antibodies detected as positive in the ELISA in these sera from patients with clinical stage I and stage II breast cancers may be induced by immunogens other than p53 protein. Many of these reactions give apparently positive signals in a variety of p53 assays, and very stringent analysis is required to avoid possible misinterpretation of these responses as a p53-specific B-cell response in human cancer patients.

Conformational changes in p53 analysed using new antibodies to the core DNA binding domain of the protein.

The p53 protein contains a protease resistant core section that binds to DNA in a sequence specific manner and whose crystal structure has been determined. This core is flanked at the N-terminus by the transcriptional transactivation domain and at the C-terminus by sequences involved in the oligomerisation of the protein. Extensive immunochemical analysis of p53 has shown that dominant antigenic sites lie within these N- and C-terminal domains while few antibodies to the central core have been identified. One of these, PAb240, has been extensively characterised as its epitope is cryptic in the native DNA binding core structure but is exposed by denaturation. This epitope is also exposed on many p53 proteins that contain point mutations in the core domain suggesting that these mutations may have a common affect on the structure of the core. To investigate this further we have generated several new antibodies to novel sites on p53 and mapped their epitopes using synthetic peptides. We find that antibodies to two other discrete sites in the core can also, like PAb240, recognize cryptic epitopes and distinguish mutant from wild-type conformations implying that the point mutations found in p53 in human tumours have widespread effects on the folding pattern of the DNA binding domain.