Immunologically defined subclasses of the growth suppressor protein p53 detected with antibodies in sera from tumor patients.

Somatic mutation of the gene encoding the cellular p53 protein is the most common event in the development of human cancer. Patients with various types of cancer have circulating antibodies against p53. We screened sera of patients with different types of tumors. Some of the sera was used to precipitate p53 from different human tumor cell lines. Serum from a patient with breast cancer recognized wild-type and mutant form of p53 from an in vitro translation reaction. p53 recognized by this serum was found to be metabolically stable and phosphorylated in vivo. Furthermore, this serum recognized p53 in a complex with a protein kinase, which phosphorylated p53 in an in vitro phosphorylation reaction.

Mutation and expression of the p53 tumor suppressor gene in tumor samples and cancer cell-lines - comparison of nonisotopic direct DNA-sequencing, immunoblotting and immunohistochemistry.

Mutations in the nuclear phosphoprotein p53 are the most frequent genetic alterations in human solid tumors detected so far. These mutations are clustered in highly conserved domains spanning from exon 4 to 9 of the gene. A very precise method of detecting p53 mutations is to sequence these domains. However, 2 to 3 overlapping PCR-amplifications were needed to span the whole mutation-prone region. We used a very rapid non radioactive solid-phase DNA sequencing method starting from mRNA to sequence the p53 domains in both directions with T7 DNA-polymerase allowing detection of the heterozygous state, where one allele shows the wild-type sequence, the other a mutated one. First we sequenced four colon carcinoma cell lines with known p53 mutations and one T-cell-leukemia cell line with a heterozygous situation to validate our method. Using this method we sequenced the p53 gene (exons four to nine) from 16 primary colon carcinomas. Seven of these 16 (44%) carcinomas showed mutations in the p53 gene resulting in amino acid exchanges. One showed a silent mutation, another one showed two point mutations in the highly conserved domain of the p53 gene. These colorectal carcinomas have been examined for overexpression of the p53 protein using a panel of monoclonal antibodies directed against p53 (PAb1801, PAb240, PAb421, PAb1620) by immunohistochemical analysis and immunoblotting. Furthermore, four colorectal cancer cell lines were examined by indirect immunofluorescence technique with the same mAb PAb1801 as used in histological staining. Analysis of 6 out of 15 (40%) tumor specimens revealed markedly positive p53 nuclear staining patterns using monoclonal antibody PAb1801. These data suggest that there is quite a good correlation between point mutation of the p53 gene and nuclear staining with monoclonal antibody PAb1801 detecting overexpressed p53 protein. Moreover, there is no convincing evidence that wild-type protein can be detected using the monoclonal antibodies PAb 1801 and PAb 1620.