Precise characterisation of monoclonal antibodies to the C-terminal region of p53 protein using the PEPSCAN ELISA technique and a new non-radioactive gel shift assay.
The development of human cancers is frequently associated with inactivation of the p53 tumour suppressor protein triggering cell cycle arrest or apoptosis in response to cellular stress. The p53 protein has been identified as a transcription factor with sequence-specific DNA binding properties. The DNA-binding activity is cryptic but can be modulated through the C-terminal region of the p53 protein by several different stimuli, including phosphorylation by casein kinase II (CKII), protein kinase C (PKC) or binding of the C-terminal monoclonal antibody PAb421. Monoclonal antibodies to the C-terminal region of p53 protein are able to activate the latent form of p53 and induce binding to DNA. To characterise such antibodies, we used a combination of the PEPSCAN ELISA procedure and a newly developed non-radioactive gel shift assay. Monoclonal antibodies from the Bp53 series displayed higher affinities for the human, rat and mouse p53 proteins than did the conventional antibody PAb421. In addition, these antibodies were able to activate the sequence-specific DNA binding functions in latent forms of p53 protein and, in contrast to PAb421, they were able to recognise both PKC phosphorylated and PKC non-phosphorylated forms of p53 protein. Our monoclonal antibodies recognising post-translationally modified target epitopes in the C-terminal region of p53 protein might assist the development of more effective molecules for p53-based cancer therapy.
Pospísilová
S
S
Department of Cellular and Molecular Oncology, Masaryk Memorial Cancer Institute, Zlutý kopec 7, 656 53, Brno, Czech Republic.
Brázda
V
V
Amrichová
J
J
Kamermeierová
R
R
Palecek
E
E
Vojtesek
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000915:Antibody Affinity; D001483:Base Sequence; D002461:Cell Line, Transformed; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018604:Epitope Mapping; D016158:Genes, p53; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000628:therapy; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D016159:Tumor Suppressor Protein p53; D004247:DNA; D011493:Protein Kinase C