title: hnRNP C1/C2 and Pur-beta proteins mediate induction of senescence by oligonucleotides homologous to the telomere overhang.
abstract: BACKGROUND
  Experimental disruption of the telomere overhang induces a potent DNA damage response and is the target of newly emerging cancer therapeutics. Introduction of T-oligo, an eleven-base oligonucleotide homologous to the 3'-telomeric overhang, mimics telomere disruption and induces DNA damage responses through activation of p53, p73, p95/Nbs1, E2F1, pRb, and other DNA damage response proteins. ATM (ataxia telangiectasia mutated) was once thought to be the primary driver of T-oligo-induced DNA damage responses; however, recent experiments have highlighted other key proteins that may also play a significant role.
  
  
  METHODS
  To identify proteins associated with T-oligo, MM-AN cells were treated with biotinylated T-oligo or complementary oligonucleotide, cell lysates were run on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and the protein bands observed after treatment of cells with T-oligo or complementary oligonucleotide were analyzed using mass spectrometry. To study the effect of T-oligo on expression of hnRNP C1/C2 (heterogeneous nuclear ribonucleoprotein C1 and C2) and purine-rich element binding proteins (Pur proteins), cells were treated with T-oligo, and immunoblotting experiments were performed. To determine their role in senescence, cells were treated with shRNA (short hairpin ribonucleic acid) against these proteins, and senescence was studied using the senescence associated beta-galactosidase assay.
  
  
  RESULTS
  Using mass spectrometry, RNA-binding hnRNP C1/C2 and DNA-binding Pur proteins were found to associate with T-oligo. hnRNP C1/C2 exhibited increased expression (3.6-12.0-fold) in non-small-cell lung cancer (NSCLC) and in melanoma cells (4.5-5.2-fold), and Pur proteins exhibited increased expression of 2.2-fold in NSCLC and 2.0-fold in melanoma cells after T-oligo treatment. Experimental knockdown of hnRNP C1/C2 and Pur-beta completely abrogated T-oligo induced senescence in both MU melanoma and H358 NSCLC cells. Additionally, knockdown of Pur-beta prevented T-oligo-induced phosphorylation of p53, hypophosphorylation of pRb, and upregulation of E2F1, p21, and p53.
  
  
  CONCLUSIONS
  These novel findings highlight proteins essential to T-oligo's anticancer effects that may be of interest in telomere biology and cancer therapeutics.
authors.affiliation: Department of Biomedical Sciences, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Mulnix
authors.affiliation: Department of Biomedical Sciences, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Pitman
authors.affiliation: College of Medicine, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Retzer
authors.affiliation: Department of Biomedical Sciences, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Ceyda
authors.initials: C
authors.lastname: Bertram
authors.affiliation: College of Medicine, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Kavin
authors.initials: K
authors.lastname: Arasi
authors.affiliation: College of Medicine, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Crees
authors.affiliation: College of Medicine, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Girard
authors.affiliation: Department of Biomedical Sciences, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Srijayaprakash B
authors.initials: SB
authors.lastname: Uppada
authors.affiliation: Department of Biomedical Sciences, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Stone
authors.affiliation: Department of Biomedical Sciences, University of Illinois at Chicago, Rockford, IL, USA.
authors.forename: Neelu
authors.initials: N
authors.lastname: Puri
keywords: NSCLC; T-oligo; melanoma; p21; p53; pRb