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.
Highlights
G-quadruplex forming DNA of gene promoter associated with cell death and growth arrest
Cells and Cell Culture—Human histiocytic lymphoma U937 cells (ATCC) and human colon carcinoma HCT 116 p53ϩ/ϩ, HCT116 p53ϩ/Ϫ, and HCT p53Ϫ/Ϫ cells were bought from the Core Cell Center of Johns Hopkins University, ATM mouse wild type, and null (334 ATMϩ/ϩ, 4a ATMϩ/ϩ, and 695 Ϯ 743Ϫ/Ϫ) cells, and lung adenocarcinoma cell lines A549 and Sk-Lu-1 were maintained in RPMI or DMEM supplemented with 10% FCS. 5 ϫ 104 cells were plated in 6-well plates and treated with 10 M of Pu-27 or control cytosine-rich oligonucleotide (CRO) day
There were no changes of DNA repair molecules (H2AX and BRCA1) and telomere maintenance gene telomerase reverse transcriptase (Fig. 6A) in U937 cells. ⌬B-U937 cells, comparing to U937 cells presented no changes of telomeric shelterin proteins (TRF2, TRF1, POT1, and TIN2), DNA damage response mediators (RAD17 and RAD50) and double-stranded breaks (DSBs) repair factor 53BP1; down-regulation of upstream kinase ATM; upregulation of G2 checkpoint molecules (CHK1 and CHK2); and DNA repair molecules (H2AX and BRCA1) (Fig. 6B)
Summary
G-quadruplex forming DNA of gene promoter associated with cell death and growth arrest. Results: G-quadruplex forming DNA at c-Myc promoter Pu27 destabilizes proteins at telomere and inhibits DNA repair molecules. 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 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. A substantial proportion (ϳ80%) of c-Myc expression is regulated by the nuclease hypersensitivity element III1 element, of which Pu-27 is a critical part An oligonucleotide encoding this sequence, Pu-27, has been shown to inhibit the proliferation of tumor cells derived from different tissues including breast, lung, and blood. The TRF2 N-terminal domain, which includes the basic domain of TRF2, condenses the telomeric end by
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