title: A pool of peptides extracted from wheat bud chromatin inhibits tumor cell growth by causing defective DNA synthesis.
abstract: BACKGROUND
  We previously reported that a pool of low molecular weight peptides can be extracted by alkali treatment of DNA preparations obtained from prokaryotic and eukaryotic cells after intensive deproteinization. This class of peptides, isolated from wheat bud chromatin, induces growth inhibition, DNA damage, G2 checkpoint activation and apoptosis in HeLa cells. In this work we studied their mechanism of action by investigating their ability to interfere with DNA synthesis.
  
  
  METHODS
  BrdUrd comet assays were used to detect DNA replication defects during S phase. DNA synthesis, cell proliferation, cell cycle progression and DNA damage response pathway activation were assessed using 3H-thymidine incorporation, DNA flow cytometry and Western blotting, respectively.
  
  
  RESULTS
  BrdUrd labelling close to DNA strand discontinuities (comet tails) detects the number of active replicons. This number was significantly higher in treated cells (compared to controls) from entry until mid S phase, but markedly lower in late S phase, indicating the occurrence of defective DNA synthesis. In mid S phase the treated cells showed less 3H-thymidine incorporation with respect to the controls, which supports an early arrest of DNA synthesis. DNA damage response activation was also shown in both p53-defective HeLa cells and p53-proficient U2OS cells by the detection of the phosphorylated form of H2AX after peptide treatment. These events were accompanied in both cell lines by an increase in p21 levels and, in U2OS cells, of phospho-p53 (Ser15) levels. At 24 h of recovery after peptide treatment the cell cycle phase distribution was similar to that seen in controls and CDK1 kinase accumulation was not detected.
  
  
  CONCLUSIONS
  The data reported here show that the antiproliferative effect exhibited by these chromatin peptides results from their ability to induce genomic stress during DNA synthesis. This effect seems to be S-phase specific since surviving cells are able to progress through their normal cell cycle when the peptide fraction is removed from the culture medium. It is likely that the subsequent apoptosis is a consequence of the failed attempt of the tumour cells to repair the DNA damage induced by the peptides.
authors.affiliation: Department of Cellular and Environmental Biology, University of Perugia via Pascoli, 06123, Perugia, Italy.
authors.forename: Loretta
authors.initials: L
authors.lastname: Mancinelli
authors.affiliation: Department of Cellular and Environmental Biology, University of Perugia via Pascoli, 06123, Perugia, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Secca
authors.affiliation: Clinic for Diagnostics and Intervention, Oslo University Hospital-Rikshospitalet, Oslo, Norway.
authors.forename: Paula M
authors.initials: PM
authors.lastname: De Angelis
authors.affiliation: Department of Cellular and Environmental Biology, University of Perugia via Pascoli, 06123, Perugia, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Mancini
authors.affiliation: Department of Clinical and Experimental Medicine, University of Perugia, Faculty of Medicin, S.Andrea delle Fratte, 06132, Perugia, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Marchesini
authors.affiliation: Department of Clinical and Experimental Medicine, University of Perugia, Faculty of Medicin, S.Andrea delle Fratte, 06132, Perugia, Italy.
authors.forename: Cristiano
authors.initials: C
authors.lastname: Marinelli
authors.affiliation: Department of Cellular and Environmental Biology, University of Perugia via Pascoli, 06123, Perugia, Italy.
authors.forename: Lanfranco
authors.initials: L
authors.lastname: Barberini
authors.affiliation: Department of Clinical and Experimental Medicine, University of Perugia, Faculty of Medicin, S.Andrea delle Fratte, 06132, Perugia, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Grignani
keywords: BrdUrd comet; Chromatin peptides; DNA damage; G2 checkpoint; H2AX
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