Abstract 2123: ATM mediated homeostasis of ‘shelterin’ at telomere is compromised by G-quadruplex leading to DNA damage

Abstract

Abstract Quadruplex-forming DNA sequences are present throughout the genome, including in the telomeres. We have recently shown that the c-myc promoter quadruplex-forming sequence, Pu-27 inhibits growth of malignant cells. Telomere integrity is maintained by the ‘shelterin’ protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks. ATM has been shown to play a role in the maintenance of telomere integrity. U937, histiocytic lymphoma cells were sensitive to Pu-27 as evident by MTT and colony formation assay and frequently showed chromosomal aberrations including abnormal metaphases (45%), chromatid breaks (40%) chromosomal breaks (27%). RT-PCR array of Pu-27 treated U937 cells revealed downregulation of ‘shelterin’ proteins TRF2, TRF1, POT1 and TIN2; induction of DNA damage response sensor γ-H2AX; downregulation of upstream kinase ATM and other downstream DNA damage mediator RAD17, RAD50, BRCA1, 53BP1, CHK1, CHK2. To understand the specific role of ‘shelterin’ protein complex, we stably transfected U937 cells with a vector expressing a dominant negative mutant of a shelterin protein TRF2 (dn-U937). These dn-UP37 cells became relatively resistant when treated with Pu-27 and showed no changes of basal level expression of ‘shelterin’ protein complex but induction of γ-H2AX and DNA repair inducing molecules BRCA1 and CHK1. This suggests that intact TRF2 is required for Pu-27 mediated U937 cellular sensitivity and it may acted by altering the telomere-‘shelterin’ complex. This idea was further substantiated by the fact that ALT (Alternative Lengthening of Telomere) cells were also relatively resistant to Pu27. To elucidate the specific role of ATM, we have shown by MTT and colony formation assay that ATM-deficient cells are more sensitive to growth inhibition by Pu-27 whereas ATM-proficient cells were relatively resistant to Pu-27. Co-immunostaining with γ-H2AX and TRF1 revealed that the sensitivity to Pu-27 in ATM deficient cells was associated with phosphorylation of H2AX, an indicator of DNA double strand break and induction of TIFs (Telomere-dysfunction Induced Foci). There was induction of γ-H2AX and no alteration of basal level of ‘shelterin’ protein complex in Pu-27 treated ATM deficient cells (as determined by RT-PCR array). A549 cells, which were sensitive to Pu-27, also showed abundant presence of TIF compared to untreated cells. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, these data suggest that downregulation of ATM destabilizes telomere-shelterin homeostasis at telomere ends in response to Pu-27 of U937 cells. Continuous exposure of G-quadruplex results in refractory DNA damage repair leading to uncompensated genomic stability and cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2123. doi:1538-7445.AM2012-2123