Abstract 743: Pu-27 G-quadruplex induces uncompensated DNA damage response by destabilizing shelterin complex at telomere and non-telomeric region leading to cell death.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Throughout the genome as well as telomere region, there is presence of DNA sequences which forms quadruplex. Here we show Pu-27 triggers the damage of cells by interfering telomeric ‘shelterin’ protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks (DSB). We found that Pu-27 frequently prompted chromosomal aberrations including abnormal metaphases (45%), chromatid breaks (40%) chromosomal break (27%) by karyotyping of U937 metaphase cells and was associated with induction of DNA damage response sensor γ-H2AX. Analysis of DNA damage response regulators by RT-PCR array of Pu-27 treated U937 cells revealed downregulation of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), upstream kinase ATM, DNA damage response mediators (RAD17, RAD50 and 53BP1), and cell cycle arrest molecules (CHK1 and CHK2). Interestingly, there were no changes or low expression of DNA repair molecules (H2AX and BRCA1) and telomere maintenance gene TERT respectively. αB-U937 cell, where basic-domain of shelterin protein TRF2 was deleted became relatively resistance to Pu-27 and showed no changes of constitutively active form of γ-H2AX. RT-PCR array of Pu-27 treated αB-U937 cell appeared to be no changes of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), DNA damage response mediators (RAD17, RAD50 and 53BP1); down regulation of upstream kinase ATM; up regulation of cell cycle arrest molecules (SMC1, CHK1 and CHK2), DNA repair molecules (H2AX and BRCA1), and telomere maintenance gene TERT. Shelterin protein, TRF1 co-associated with γH2AX by inducing TIFs (Telomere-dysfunction Induced Foci) when U937 cell was treated with Pu-27. Sk-Lu-1, an Alt (Alternating Lengthening to Telomere) cell which does not use TERT for their growth and propagation is relatively resistance to Pu-27. ATM preserves the telomeric homeostasis by maintaining the integrity of ‘shelterin’ protein. To substantiate the specificity of Pu-27 to shelterin protein complex, we found ATM-deficient cell was more sensitive to Pu-27 than ATM-proficient cell. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, these results suggest that Pu-27-treated sensitivity at least in part due to compromising telomeric shelterin complex and perhaps stabilizing telomeric quadruplex and not by directly inhibiting telomerase enzyme. U937 cell with chromosomal aberration and unstable telomere progress through cell cycle without arrest and repair leading to cell death whereas αB-U937 cells is continuously repaired by arresting and induction of repair machinery. Continuous exposure of Pu-27 results in refractory to repair of DNA damage leading to uncompensated genomic stability leading to cell death. Citation Format: Md Ashraful Islam, Shelia D. Thomas, Kara J. Sedoris, Donald M. Miller. Pu-27 G-quadruplex induces uncompensated DNA damage response by destabilizing shelterin complex at telomere and non-telomeric region leading to cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2013-743 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.