Françoise Hoffschir

Apoptosis related to telomere instability and cell cycle alterations in human glioma cells treated by new highly selective G-quadruplex ligands

Telomerase represents a relevant target for cancer therapy. Molecules able to stabilize the G-quadruplex (G4), a structure adopted by the 3′-overhang of telomeres, are thought to inhibit telomerase by blocking its access to telomeres. We investigated the cellular effects of four new 2,6-pyridine-dicarboxamide derivatives displaying strong selectivity for G4 structures and strong inhibition of telomerase in in vitro assays. These compounds inhibited cell proliferation at very low concentrations and then induced a massive apoptosis within a few days in a dose-dependent manner in cultures of three telomerase-positive glioma cell lines, T98G, CB193 and U118-MG. They had also antiproliferative effects in SAOS-2, a cell line in which telomere maintenance involves an alternative lengthening of telomeres (ALT) mechanism. We show that apoptosis was preceded by multiple alterations of the cell cycle: activation of S-phase checkpoints, dramatic increase of metaphase duration and cytokinesis defects. These effects were not associated with telomere shortening, but they were directly related to telomere instability involving telomere end fusion and anaphase bridge formation. Pyridine-based G-quadruplex ligands are therefore promising agents for the treatment of various tumors including malignant gliomas.

Preferential binding of a G-quadruplex ligand to human chromosome ends

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, 3H-360A (2,6-N,N′-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-3H]. We verified the in vitro selectivity of 3H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that 3H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3′-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with 3H-360A. We found that 3H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.

Alternative Lengthening of Telomeres in Human Glioma Stem Cells

Cancer stem cells are increasingly recognized as major therapeutic targets. We report here the isolation of glioma stem cells (GSCs) maintaining telomere length through a telomerase‐independent mechanism known as alternative lengthening of telomeres (ALTs). TG20 cells were isolated from a glioblastoma multiforme, which had the ALT phenotype. They have no detectable telomerase activity and extremely long and heterogeneous telomeres colocalizing with promyelocytic leukemia bodies. The cancer stem cell potential of TG20 cells was confirmed based on their expression of neural stem cell markers, their capacity of in vitro long‐term proliferation and to form intracranial tumors in immune‐deficient mice. Interestingly, we found that both in vitro and in vivo TG20 cells were significantly more resistant to ionizing radiation than GSCs with telomerase activity. Analysis of DNA damage foci, DNA double‐strand breaks repair, and chromosome instability suggest that radiation resistance was related to interference of ALT pathway with DNA damage response. Therefore, our data show for the first time that the ALT pathway can confer to cancer stem cells the capacity to sustain long‐term proliferation as telomerase activity and importantly may also affect treatment efficiency. TG20 cells are thus the first cellular model of GSCs displaying ALT and should prove to be useful for the development of specific treatment strategies. STEM CELLS 2011;29:440–451

Persistent oxidative stress after ionizing radiation is involved in inherited radiosensitivity

The SW620IR1 cell line was derived from SW620 human colon cells surviving to ionizing radiations. It shows an increased radiosensitivity and a higher yield of spontaneous chromosomal aberrations. In order to check whether altered reactive oxygen intermediates (ROI) metabolism is involved in this inherited phenotype, we compared the two cell lines for their radiation-induced modifications at the level of ROI production, antioxidant activities, and chromosomal aberrations. Compared to SW620, SW620IR1 cells exhibit a higher and more persistent ROI induction after various doses of ionizing radiations and a higher yield of dicentric chromosomes. They are also characterized by lower basal activities of glutathione peroxidase and manganese-containing superoxide dismutase, and lower ability to induce these antioxidant defenses after irradiation. Resumption of cell growth after irradiation coincides with maximal induction of antioxidant activities and normalization of ROI concentration. However, at that time radiation-induced chromosomal aberrations are not completely eliminated, leading to the proliferation of genetically unstable cells. These results indicate that the inherited sensitivity of SW620IR1 cells is associated with altered antioxidant activities resulting in higher and more prolonged oxidative stress after radiation exposure. They also suggest that the normalization of ROI levels allows these p53 mutant cells to resume proliferation although high levels of DNA damages are still persisting, thereby explaining the chromosomal instability observed as a delayed effect of radiation exposure.

Low Catalase Activity in Xeroderma Pigmentosum Fibroblasts and SV40-Transformed Human Cell Lines is Directly Related to Decreased Intracellular Levels of the Cofactor, NADPH

We have previously shown that fibroblasts from ultra-violet (UV) hypersensitive xeroderma pigmentosum patients (XP) are markedly deficient in catalase activity resulting in high intracellular levels of hydrogen peroxide (H2O2) following UV irradiation. No direct correlation between catalase activity and repair ability was found since XP variant cells which are proficient in nucleotide excision repair (NER) showed activities as low as those found in NER deficient classical XP groups A and D. However, in contrast to the skin cancer prone XP patients, another NER deficient syndrome, trichothiodystrophy (TTD), which does not exhibit any cancer predisposition, was found to present normal catalase activity. Moreover, it was found that a variety of SV40 transformed human cell lines also showed decreased catalase activities. Our previous data showed that a molecular analysis of the normal, XP, TTD or transformed human fibroblast cell lines did not reveal any differences in levels of catalase transcription or amount of catalase protein subunits. These results incited us to examine the structure/function relationship of the tetrameric active enzyme form of catalase (which is the only one able to carry out H2O2 dismutation) with its cofactor NADPH. In the present study, we have measured the effects on catalase activity after adding NADPH either to acellular extracts or during cell culture of the different cell types. The NADPH levels were also quantified directly in intact cells using flow cytometry. Our results show a clear relationship between low catalase activity and striking decrease in intracellular NADPH levels.

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors.

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca−/− and fancg−/− mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca−/− and fancg−/− mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self‐renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.

ATR contributes to telomere maintenance in human cells.

Telomere maintenance is essential to preserve genomic stability and involves several telomere-specific proteins as well as DNA replication and repair proteins. The kinase ATR, which has a crucial function in maintaining genome integrity from yeast to human, has been shown to be involved in telomere maintenance in several eukaryotic organisms, including yeast, Arabidopsis and Drosophila. However, its role in telomere maintenance in mammals remains poorly explored. Here, we report by using telomere-fluorescence in situ hybridization (Telo-FISH) on metaphase chromosomes that ATR deficiency causes telomere instability both in primary human fibroblasts from Seckel syndrome patients and in HeLa cells. The telomere aberrations resulting from ATR deficiency (i.e. sister telomere fusions and chromatid-type telomere aberrations) are mainly generated during and/or after telomere replication, and involve both leading and lagging strand telomeres as shown by chromosome orientation-FISH (CO-FISH). Moreover, we show that ATR deficiency strongly sensitizes cells to the G-quadruplex ligand 360A, enhancing sister telomere fusions and chromatid-type telomere aberrations involving specifically the lagging strand telomeres. Altogether, these data reveal that ATR plays a critical role in telomere maintenance during and/or after telomere replication in human cells.

Role of Fanconi DNA repair pathway in neural stem cell homeostasis.

Defects in DNA repair pathways have been involved in collapse of early neurogenesis leading to brain development abnormalities and embryonic lethality. However, consequences of DNA repair defects in adult neural stem and progenitor cells and their potential contribution in ageing phenotype are poorly understood. The Fanconi anaemia (FA) pathway, which functions primarily as a DNA damage response system, has been examined in neural stem and progenitor cells during developmental and adult neurogenesis. We have shown that loss of fanca and fancg specifically provokes neural progenitor apoptosis during forebrain development, related to DNA repair defects, which persists in adulthood leading to depletion of the neural stem cell pool with ageing. In addition, neural stem cells from FA mice had a reduced capacity to self-renew in vitro. Here, we expand upon our recent work and give further data examining possible implication of oxidative stress. Therefore, FA phenotype might be interpreted as a premature ageing of stem cells, DNA damages being among the driving forces of ageing.

Rad51 and DNA-PKcs are involved in the generation of specific telomere aberrations induced by the quadruplex ligand 360A that impair mitotic cell progression and lead to cell death

Functional telomeres are protected from non-homologous end-joining (NHEJ) and homologous recombination (HR) DNA repair pathways. Replication is a critical period for telomeres because of the requirement for reconstitution of functional protected telomere conformations, a process that involves DNA repair proteins. Using knockdown of DNA-PKcs and Rad51 expression in three different cell lines, we demonstrate the respective involvement of NHEJ and HR in the formation of telomere aberrations induced by the G-quadruplex ligand 360A during or after replication. HR contributed to specific chromatid-type aberrations (telomere losses and doublets) affecting the lagging strand telomeres, whereas DNA-PKcs-dependent NHEJ was responsible for sister telomere fusions as a direct consequence of G-quadruplex formation and/or stabilization induced by 360A on parental telomere G strands. NHEJ and HR activation at telomeres altered mitotic progression in treated cells. In particular, NHEJ-mediated sister telomere fusions were associated with altered metaphase-anaphase transition and anaphase bridges and resulted in cell death during mitosis or early G1. Collectively, these data elucidate specific molecular and cellular mechanisms triggered by telomere targeting by the G-quadruplex ligand 360A, leading to cancer cell death.

Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells

DNA ligase I (LigI) plays a central role in the joining of strand interruptions during replication and repair. In our current study, we provide evidence that DNA ligase III (LigIII) and XRCC1, which form a complex that functions in single-strand break repair, are required for the proliferation of mammalian LigI-depleted cells. We show from our data that in cells with either dysfunctional LigI activity or depleted of this enzyme, both LigIII and XRCC1 are retained on the chromatin and accumulate at replication foci. We also demonstrate that the LigI and LigIII proteins cooperate to inhibit sister chromatid exchanges but that only LigI prevents telomere sister fusions. Taken together, these results suggest that in cells with dysfunctional LigI, LigIII contributes to the ligation of replication intermediates but not to the prevention of telomeric instability.