No abstract

Cells and viruses. CeUs were grown as previously described (8). The SV40-transformed mouse cells (SVA31E7 and VLM) were kindly provided by Y. Ito and S. Tevethia, respectively; the SV40-transformed rat cells (14B), by D. Lane; the SV40-transformed hamster cells (H65 90B), by S. Tevethia; the SV40transformed rabbit cells (TRK54), by P. Black; the SV40-transformed human cells (SV80), by E. Gurney; and the methylcholanthrene-transformed mouse cells (L929), by D. Cox. All viruses were propagated on CV-1 African green monkey kidney cells as described previously (8). The SV-S strain was used as wild-type 861

We analyzed p53 cDNA and genomic clones from a variety of normal and transformed cells. Sequence analysis of these clones revealed that amino acid residue 72 can be an arginine, proline, or cysteine. This single codon difference results in electrophoretically distinct forms of human p53 seen in normal and transformed cells.

Antibodies reacting with the host protein p53 were found in the sera of patients with primary or secondary carcinoma of the breast. Fourteen out of the 155 sera from breast cancer patients tested were positive for anti-p53 antibodies (9%) and no positives were detected among 164 control sera from normal women tested. The locations of the first metastasis in patients with positive sera were unusual, with more lung metastases and fewer bone metastases than expected. The detection of anti-p53 antibodies indicates that p53 is altered in amount, type or presentation in breast tumors so that it becomes immunogenic.

The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti-(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos-1 cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with j-galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognised by the antibodies. At least two previously unrecognised epitopes are involved and two of the antibodies are human-p53-specific. The epitopes are denaturation-resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme-linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos-I) cells cause expression of human p53 recognised by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.