DNA damage signaling induced by the G-quadruplex ligand 12459 is modulated by PPM1D/WIP1 phosphatase.

The triazine derivative 12459 is a potent G-quadruplex ligand that triggers apoptosis or delayed growth arrest, telomere shortening and G-overhang degradation, as a function of its concentration and time exposure to the cells. We have investigated here the DNA damage response induced by 12459 in A549 cells. Submicromolar concentrations of 12459 triggers a delayed Chk1-ATR-mediated DNA damage response associated with a telomeric dysfunction and a G2/M arrest.

Mitochondrial topoisomerase I is critical for mitochondrial integrity and cellular energy metabolism.

Background: Mitochondria contain their own DNA genome (mtDNA), as well as specific DNA replication and protein synthesis machineries. Relaxation of the circular, double-stranded mtDNA relies on the presence of topoisomerase activity. Three different topoisomerases have been identified in mitochondria: Top1mt, Top3α and a truncated form of Top2β.

Coordinated regulation of mitochondrial topoisomerase IB with mitochondrial nuclear encoded genes and MYC.

Mitochondrial DNA (mtDNA) is entirely dependent on nuclear genes for its transcription and replication. One of these genes is TOP1MT, which encodes the mitochondrial DNA topoisomerase IB, involved in mtDNA relaxation. To elucidate TOP1MT regulation, we performed genome-wide profiling across the 60-cell line panel (the NCI-60) of the National Cancer Institute Developmental Therapeutics Program.

Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks.

Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1).

Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres.

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences.

Targeting telomeres and telomerase.

Telomeres and telomerase represent, at least in theory, an extremely attractive target for cancer therapy. The objective of this review is to present the latest view on the mechanism(s) of action of telomerase inhibitors, with an emphasis on a specific class of telomere ligands called G-quadruplex ligands, and to discuss their potential use in oncology.

Telomestatin-induced telomere uncapping is modulated by POT1 through G-overhang extension in HT1080 human tumor cells.

Telomestatin is a potent G-quadruplex ligand that interacts with the 3' telomeric overhang, leading to its degradation, and induces a delayed senescence and apoptosis of cancer cells. POT1 and TRF2 were recently identified as specific telomere-binding proteins involved in telomere capping and t-loop maintenance and whose interaction with telomeres is modulated by telomestatin. We show here that the treatment of HT1080 human tumor cells by telomestatin induces a rapid decrease of the telomeric G-overhang and of the double-stranded telomeric repeats.

The G-quadruplex ligand telomestatin inhibits POT1 binding to telomeric sequences in vitro and induces GFP-POT1 dissociation from telomeres in human cells.

Telomestatin is a potent G-quadruplex ligand that specifically interacts with the 3' telomeric overhang, leading to its degradation and that induces a delayed senescence and apoptosis of cancer cells. Protection of Telomere 1 (POT1) was recently identified as a specific single-stranded telomere-binding protein involved in telomere capping and T-loop maintenance. We showed here that a telomestatin treatment inhibits POT1 binding to the telomeric overhang in vitro.

Overexpression of Bcl-2 is associated with apoptotic resistance to the G-quadruplex ligand 12459 but is not sufficient to confer resistance to long-term senescence.

The triazine derivative 12459 is a potent G-quadruplex interacting agent that inhibits telomerase activity. This agent induces time- and dose-dependent telomere shortening, senescence-like growth arrest and apoptosis in the human A549 tumour cell line. We show here that 12459 induces a delayed apoptosis that activates the mitochondrial pathway.

Resistance to senescence induction and telomere shortening by a G-quadruplex ligand inhibitor of telomerase.

The molecular mechanisms induced by G-quadruplex ligands to trigger senescence in mammalian cells are still unknown, although the critical role of telomerase is highly suspected. JFA2 cells selected for resistance to senescence induced by the G-quadruplex ligand 12459 presented an overexpression of hTERT transcript that correlated to a functional increase in telomerase activity and telomere length. Consistently, treatment with 12459 failed to trigger senescence and telomere shortening in JFA2 cells.

Resistance to the short term antiproliferative activity of the G-quadruplex ligand 12459 is associated with telomerase overexpression and telomere capping alteration.

Ligands that stabilize the telomeric G-rich single-stranded DNA overhang into G-quadruplex can be considered as potential antitumor agents that block telomere replication. Ligand 12459, a potent G-quadruplex ligand that belongs to the triazine series, has been previously shown to induce both telomere shortening and apoptosis in the human A549 cell line as a function of its concentration and time exposure. We show here that A549 clones obtained after mutagenesis and selected for resistance to the short term effect of ligand 12459 frequently displayed hTERT transcript overexpression (2-6-fold).