c-Myc quadruplex-forming sequence Pu-27 induces extensive damage in both telomeric and nontelomeric regions of DNA

J Biol Chem. 2014 Mar 21;289(12):8521-31. doi: 10.1074/jbc.M113.505073. Epub 2014 Jan 24.

Abstract

Quadruplex-forming DNA sequences are present throughout the eukaryotic genome, including in telomeric DNA. We have shown that the c-Myc promoter quadruplex-forming sequence Pu-27 selectively kills transformed cells (Sedoris, K. C., Thomas, S. D., Clarkson, C. R., Muench, D., Islam, A., Singh, R., and Miller, D. M. (2012) Genomic c-Myc quadruplex DNA selectively kills leukemia. Mol. Cancer Ther. 11, 66-76). In this study, we show that Pu-27 induces profound DNA damage, resulting in striking chromosomal abnormalities in the form of chromatid or chromosomal breaks, radial formation, and telomeric DNA loss, which induces γ-H2AX in U937 cells. Pu-27 down-regulates telomeric shelterin proteins, DNA damage response mediators (RAD17 and RAD50), double-stranded break repair molecule 53BP1, G2 checkpoint regulators (CHK1 and CHK2), and anti-apoptosis gene survivin. Interestingly, there are no changes of DNA repair molecules H2AX, BRCA1, and the telomere maintenance gene, hTERT. ΔB-U937, where U937 cells stably transfected with deleted basic domain of TRF2 is partially sensitive to Pu-27 but exhibits no changes in expression of shelterin proteins. However, there is an up-regulation of CHK1, CHK2, H2AX, BRCA1, and survivin. Telomere dysfunction-induced foci assay revealed co-association of TRF1with γ-H2AX in ATM deficient cells, which are differentially sensitive to Pu-27 than ATM proficient cells. Alt (alternating lengthening of telomere) cells are relatively resistant to Pu-27, but there are no significant changes of telomerase activity in both Alt and non-Alt cells. Lastly, we show that this Pu-27-mediated sensitivity is p53-independent. The data therefore support two conclusions. First, Pu-27 induces DNA damage within both telomeric and nontelomeric regions of the genome. Second, Pu-27-mediated telomeric damage is due, at least in part, to compromise of the telomeric shelterin protein complex.

Keywords: ATM; Alt Cell; Cell Death; DNA Damage; G-quadruplex; Myc; Phospho-H2AX; Shelterin; Survivin; Telomeres.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins / genetics
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Base Sequence
  • Cell Death
  • Cell Line, Tumor
  • DNA / chemistry
  • DNA / genetics*
  • DNA / metabolism
  • DNA Damage*
  • G-Quadruplexes*
  • Genes, myc*
  • Histones / genetics
  • Histones / metabolism
  • Humans
  • Neoplasms / genetics*
  • Neoplasms / metabolism
  • Neoplasms / pathology
  • Phosphorylation
  • Promoter Regions, Genetic
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism
  • Shelterin Complex
  • Telomere / chemistry
  • Telomere / genetics*
  • Telomere / metabolism
  • Telomere-Binding Proteins / genetics
  • Telomere-Binding Proteins / metabolism
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • H2AX protein, human
  • Histones
  • Proto-Oncogene Proteins c-myc
  • Shelterin Complex
  • Telomere-Binding Proteins
  • Tumor Suppressor Protein p53
  • DNA
  • Ataxia Telangiectasia Mutated Proteins