Co-localization of endogenous and exogenous p53 proteins in nasopharyngeal carcinoma cells.
Recently, we have established nine nasopharyngeal carcinoma (NPC) cell lines in which only one cell line showed the p53 mutation. For investigation of the p53 mutation in this line, immunostaining using anti-p53 antibody was applied and showed the presence of p53 protein in the cytoplasm but not in the nucleus. Single strand conformation polymorphism analysis of the p53 gene showed one normal and one additional DNA band. Cloning and sequencing of PCR-amplified DNA showed an AGA (arginine) to ACA (threonine) heterozygous point mutation at codon 280. Transfection of the p53 DNA binding sequence and chloramphenicol acetyltransferase assay revealed loss of transcriptional activation function of endogenous p53 protein. Co-localization of the endogenous and the transfected exogenous p53 protein by polyclonal antibodies to anti-p53 protein revealed strong exogenous p53 staining in the transfected nuclei and weak staining of endogenous p53 protein in the cytoplasm. We concluded that (a) a heterozygous point mutation at codon 280 was identified in the NPC-TW 06 cell line; (b) the point mutation may cause the stagnation of mutant p53 protein in the cytoplasm, and loss of its transcriptional activation function; (c) endogenous and exogenous p53 protein can be co-localized at the same time in the transfected cells; and (d) 280 mutant p53 protein in NPC cells does not cause a decrease or increase in sensitivity to chemotherapy.
Hwang
J K
JK
Institute of Pathology, College of Medicine, National Taiwan University, Taipei, R.O.C.
Lin
C T
CT
D001706:Biopsy; D015152:Blotting, Northern; D015139:Blotting, Southern; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000737:chemistry; D015500:Chloramphenicol O-Acetyltransferase; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D009303:Nasopharyngeal Neoplasms / Q000737:chemistry*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis* / Q000235:genetics / Q000276:immunology
D016159:Tumor Suppressor Protein p53; D015500:Chloramphenicol O-Acetyltransferase