title: Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function.
abstract: The p53 tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line p53 mutations cause a familial predisposition for cancer. Germ-line or sporadic p53 mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to gamma irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in p53-dependent apoptotic pathways after treatment with gamma irradiation or doxorubicin, whereas p53-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated p53 can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.
authors.affiliation: Department of Biology and Center for Cancer Research, and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Annemieke
authors.initials: A
authors.lastname: de Vries
authors.forename: Elsa R
authors.initials: ER
authors.lastname: Flores
authors.forename: Barbara
authors.initials: B
authors.lastname: Miranda
authors.forename: Harn-Mei
authors.initials: HM
authors.lastname: Hsieh
authors.forename: Conny Th M
authors.initials: CT
authors.lastname: van Oostrom
authors.forename: Julien
authors.initials: J
authors.lastname: Sage
authors.forename: Tyler
authors.initials: T
authors.lastname: Jacks
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001755:Blastocyst; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics; D004317:Doxorubicin / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D005816:Genetic Complementation Test; D006801:Humans; D006822:Hybrid Cells; D008221:Lymphoid Tissue; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D013234:Stem Cells / Q000166:cytology; D013950:Thymus Gland / Q000166:cytology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*; D051028:bcl-2-Associated X Protein
chemical_list: D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; D016159:Tumor Suppressor Protein p53; C064655:V(D)J recombination activating protein 2; D051028:bcl-2-Associated X Protein; D004317:Doxorubicin