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. A549 cell lines selected for resistance to 12459 and previously characterized for an altered hTERT expression also showed Bcl-2 overexpression. Transfection of Bcl-2 into A549 cells induced a resistance to the short-term apoptotic effect triggered by 12459, suggesting that Bcl-2 is an important determinant for the activity of 12459. In sharp contrast, the Bcl-2 overexpression was not sufficient to confer resistance to the senescence-like growth arrest induced by prolonged treatment with 12459. We also show that 12459 provokes a rapid degradation of the telomeric G-overhang in conditions that paralleled the apoptosis induction. In contrast, the G-overhang degradation was not observed when apoptosis was induced by camptothecin. Bcl-2 overexpression did not modify the G-overhang degradation, suggesting that this event is an early process uncoupled from the final apoptotic pathway

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. We show that TOP1MT mRNA expression varies widely across these cell lines with the highest levels in leukemia (HL-60, K-562) and melanoma (SK-MEL-28), intermediate levels in breast (MDA-MB-231), ovarian (OVCAR) and colon (HCT-116, HCT-15, KM-12), and lowest levels in renal (ACHN, A498), prostate (PC-3, DU-145) and central nervous system cell lines (SF-539, SF-268, SF-295). Genome-wide analyses show that TOP1MT expression is significantly correlated with the other mitochondrial nuclear-encoded genes including the mitochondrial nucleoid genes, and demonstrate an overall co-regulation of the mitochondrial nuclear-encoded genes. We also find very high correlation between the expression of TOP1MT and the proto-oncogene MYC (c-myc). TOP1MT contains E-boxes (c-myc binding sites) and TOP1MT transcription follows MYC up- and down-regulation by MYC promoter activation and siRNA against MYC. Our finding implicates MYC as a novel regulator of TOP1MT and confirms its role as a master regulator of MNEGs and mitochondrial nucleoids

Etude de ligands de l'ADN G-quadruplexe sur l'intégralité des télomères : voies des dommages à l'ADN au cours de l'apoptose et de la senescence dans des lignées tumorales humaines

REIMS-BU Santé (514542104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

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

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.The present study shows the importance of Top1mt in mitochondrial homeostasis. Here we show that Top1mt-/- murine embryonic fibroblasts (MEF) exhibit dysfunctional mitochondrial respiration, which leads decreased ATP production and compensation by increased glycolysis and fatty acid oxidation. ROS production in Top1mt-/- MEF cells is involved in nuclear DNA damage and induction of autophagy. Lack of Top1mt also triggers oxidative stress and DNA damage associated with lipid peroxidation and mitophagy in Top1mt-/- mice.Together, our data implicate Top1mt for mitochondrial integrity and energy metabolism. The compensation mechanism described here contributes to the survival of Top1mt-/- cells and mice despite alterations of mitochondrial functions and metabolism. Therefore, this study supports a novel model for cellular adaptation to mitochondrial damage

The G-quadruplex Ligand Telomestatin Inhibits POT1 Binding to Telomeric Sequences In vitro and Induces GFP-POT1 Dissociation from Telomeres in Human Cells

International audienceTelomestatin 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. The treatment of human EcR293 cells by telomestatin induces a dramatic and rapid delocalization of POT1 from its normal telomere sites but does not affect the telomere localization of the double-stranded telomere-binding protein TRF2. Thus, we propose that G-quadruplex stabilization at telomeric G-overhang inactivates POT1 telomeric function, generating a telomere dysfunction in which chromosome ends are no longer properly protected

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

Topoisomerase (Topo) IIIα 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 IIIα colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIα 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. Topo IIIα depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIα expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIα is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function