title: PIN3 duplication may be partially responsible for TP53 haploinsufficiency.
abstract: BACKGROUND
  Previously we have suggested that cancer cells develop a mechanism(s) which allows for either: silencing of the wild-type TP53 transcription, degradation of the wild-type TP53 mRNA, or selective overproduction of the mutated TP53 mRNA, which is the subject of this article. Sequencing of TP53 on the respective cDNA and DNA templates from tumor samples were found to give discordant results. DNA analysis showed a pattern of heterozygous mutations, whereas the analysis of cDNA demonstrated the mutated template only. We hypothesized that different TP53 gene expression levels of each allele may be caused by the polymorphism within intron 3 (PIN3). The aim of this study was to test if one of the polymorphic variants of PIN3 (A1 or A2) in the heterozygotes is associated with a higher TP53 expression, and therefore, responsible for the haploinsufficiency phenomenon.
  
  
  METHODS
  250 tumor samples were tested. To analyze the involvement of PIN3 polymorphic variant (A1 or A2) on TP53 mRNA expression regulation, bacterial subcloning combined with sequencing analyses, dual luciferase reporter assays and bioinformatic analysis were performed.
  
  
  RESULTS
  Haplotype analysis showed the predominance of the mutated template during the cDNA sequencing in all samples showing a heterozygous TP53 mutation and PIN3 heterozygosity. Out of 30 samples (from the total of 250 tested samples) which carried TP53 mutations and had a bias in allelic expression 6 were heterozygous for the A1/A2 polymorphism, and all 6 (p = 0.04) samples carried the mutation within the PIN3 longer allele (A2). Reporter assays revealed higher luciferase activity in cells transfected with the plasmid containing A2 construct than A1 and control. A2/A1 ratio ranged from 1.16 for AD293 cell line (p = 0.019) to 1.59 for SW962 cell line (p = 0.0019). Moreover, bioinformatic analyses showed that PIN3 duplication stabilized secondary DNA structures - G-quadruplexes.
  
  
  CONCLUSIONS
  TP53 alleles are not equivalent for their impact on the regulation of expression of TP53 mRNA. Therefore, in PIN3-heterozygous cases a single TP53 mutation of the longer allele might sufficiently destabilize its function. Secondary DNA structures such as quadruplexes can also play a role in PIN3-dependent TP53 haploinsufficiency.
authors.affiliation: Department of Tumor Biology, Medical University of Lodz, Zeligowskiego 7/9, 90-752, Lodz, Poland. marta.winiecka@gmail.com.
authors.forename: Marta
authors.initials: M
authors.lastname: Winiecka-Klimek
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Szybka
authors.forename: Piotr
authors.initials: P
authors.lastname: Rieske
authors.forename: Sylwester
authors.initials: S
authors.lastname: Piaskowski
authors.forename: Michal
authors.initials: M
authors.lastname: Bienkowski
authors.forename: Maciej
authors.initials: M
authors.lastname: Walczak
authors.forename: Marcin
authors.initials: M
authors.lastname: Pacholczyk
authors.forename: Michal
authors.initials: M
authors.lastname: Rostkowski
authors.forename: Jolanta
authors.initials: J
authors.lastname: Zieba
authors.forename: Mateusz
authors.initials: M
authors.lastname: Banaszczyk
authors.forename: Krystyna
authors.initials: K
authors.lastname: Hulas-Bigoszewska
authors.forename: Joanna
authors.initials: J
authors.lastname: Peciak
authors.forename: Rafal
authors.initials: R
authors.lastname: Pawliczak
authors.forename: Ewelina
authors.initials: E
authors.lastname: Stoczynska-Fidelus
mesh_terms: D045744:Cell Line, Tumor; D020440:Gene Duplication; D015972:Gene Expression Regulation, Neoplastic; D014644:Genetic Variation; D057895:Haploinsufficiency; D006801:Humans; D007438:Introns; D009369:Neoplasms / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53