title: Functional characterisation of p53 mutants identified in breast cancers with suboptimal responses to anthracyclines or mitomycin.
abstract: BACKGROUND
  Approximately 4300 different TP53 mutations have been reported in human cancers. TP53 mutations, in particular those affecting the L2/L3 domains, are associated with resistance to anthracycline or mitomycin treatment in breast cancer patients. While many mutations have been characterised functionally, novel TP53 mutations are continuously reported. Here, we characterise 10 p53 protein variants encoded by mutated TP53 (5 within and 5 outside L2/L3) detected in locally advanced or metastatic breast cancers. Each tumour was previously characterised for response to therapy, allowing comparison between in vivo and in vitro findings.
  
  
  METHODS
  Mutated p53 variants were analysed for their ability to oligomerise with the wild-type protein and their subcellular localisation by immunoprecipitation and immunofluorescence, respectively. Their ability to induce transcription of target genes was determined by qPCR. Cellular growth rate, apoptosis and senescence were monitored by WST-1, TUNEL and beta-galactosidase assays, respectively.
  
  
  RESULTS
  Immunoprecipitation assays revealed each mutant protein to retain binding capacity for wild-type p53, thus potentially acting in a dominant negative manner. Even though each p53 variant located predominantly in the nucleus, the percentage of cells with only nuclear p53 localisation varied between 60% and 90%. None of the p53 variants were able to induce target genes to levels similar to wild-type p53, nor where they able to reduce cellular growth rate, induce apoptosis or senescence similar to wild-type p53 after anthracycline treatment in vitro.
  
  
  CONCLUSIONS
  All the 10 variants studied displayed inferior p53 functionality compared to the wild-type protein.
  
  
  CONCLUSIONS
  Our data add further information characterising the effects of somatic TP53 mutations on p53 protein function and anthracycline resistance in breast cancer.
authors.affiliation: Section of Oncology, Institute of Medicine, University of Bergen, Jonas Lies vei 65, N-5021 Bergen, Norway.
authors.forename: Elisabet O
authors.initials: EO
authors.lastname: Berge
authors.forename: Johanna
authors.initials: J
authors.lastname: Huun
authors.forename: Johan R
authors.initials: JR
authors.lastname: Lillehaug
authors.forename: Per E
authors.initials: PE
authors.lastname: Lønning
authors.forename: Stian
authors.initials: S
authors.lastname: Knappskog
mesh_terms: D018943:Anthracyclines / Q000494:pharmacology*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001706:Biopsy; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology*; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
chemical_list: D018943:Anthracyclines; D000903:Antibiotics, Antineoplastic; D016159:Tumor Suppressor Protein p53; D016685:Mitomycin