“Mitotic Slippage” and Extranuclear DNA in Cancer Chemoresistance: A Focus on Telomeres

Mitotic slippage (MS), the incomplete mitosis that results in a doubled genome in interphase, is a typical response of TP53-mutant tumors resistant to genotoxic therapy. These polyploidized cells display premature senescence and sort the damaged DNA into the cytoplasm. In this study, we explored MS in the MDA-MB-231 cell line treated with doxorubicin (DOX). We found selective release into the cytoplasm of telomere fragments enriched in telomerase reverse transcriptase (hTERT), telomere capping protein TRF2, and DNA double-strand breaks marked by γH2AX, in association with ubiquitin-binding protein SQSTM1/p62. This occurs along with the alternative lengthening of telomeres (ALT) and DNA repair by homologous recombination (HR) in the nuclear promyelocytic leukemia (PML) bodies. The cells in repeated MS cycles activate meiotic genes and display holocentric chromosomes characteristic for inverted meiosis (IM). These giant cells acquire an amoeboid phenotype and finally bud the depolyploidized progeny, restarting the mitotic cycling. We suggest the reversible conversion of the telomerase-driven telomere maintenance into ALT coupled with IM at the sub-telomere breakage sites introduced by meiotic nuclease SPO11. All three MS mechanisms converging at telomeres recapitulate the amoeba-like agamic life-cycle, decreasing the mutagenic load and enabling the recovery of recombined, reduced progeny for return into the mitotic cycle.

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Giant cell glioblastoma is a distinctive subtype of glioma characterized by vulnerability to DNA damage

Brain Tumor Pathology ◽

10.1007/s10014-019-00355-w ◽

2019 ◽

Vol 37 (1) ◽

pp. 5-13 ◽

Cited By ~ 2

Author(s):

Kaoru Ogawa ◽

Akira Kurose ◽

Akihisa Kamataki ◽

Kenichiro Asano ◽

Kosuke Katayama ◽

...

Keyword(s):

Dna Damage ◽

Giant Cell ◽

Small Cells ◽

Wild Type ◽

Dna Double Strand Breaks ◽

Proliferating Cells ◽

Strand Breaks ◽

Mitotic Slippage ◽

Minimal Invasion ◽

Giant Cell Glioblastoma

Abstract Giant cell glioblastoma (GC-GBM) consists of large cells with pleomorphic nuclei. As a contrast to GC-GBM, we defined monotonous small GBM (MS-GBM) as GBM that consists of small cells with monotonous small nuclei, and compared the DNA damage as well as other pathological features. GC-GBM showed minimal invasion (< 2 mm) and focal sarcomatous areas. TERTp was wild type in GC-GBM but mutant in MS-GBM. OLIG2 expression was significantly higher in MS-GBM (P < 0.01) (77% in MS-GBM and 7% in GC-GBM). GC-GBM showed significantly higher DNA double-strand breaks (DSBs) compared with MS-GBM (P < 0.01) (76% in GC-GBM and 15% in MS-GBM). Nearly, all large cells in GC-GBM underwent DSBs. Thus, significant DSBs in GC-GBM might be induced by an innate lesser stemness characteristic and be followed by mitotic slippage, resulting in polyploidization and the large pleomorphic nuclei. We conclude that GC-GBM is a distinctive subtype of glioma characterized by its vulnerability to DNA damage and that wild-type TERTp and lower OLIG2 function might induce this feature. Notably, even large pleomorphic nuclei with severe DSBs demonstrated Ki67 positivity, which alerts pathologists to the interpretation of Ki67 positivity, because cells with large nuclei undergoing severe DSBs cannot be recognized as proliferating cells that contribute to tumor aggressiveness.

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Ku and the Stability of the Genome

Journal of Biomedicine and Biotechnology ◽

10.1155/s1110724302201035 ◽

2002 ◽

Vol 2 (2) ◽

pp. 61-65 ◽

Cited By ~ 2

Author(s):

Anna A. Friedl

Keyword(s):

Double Strand Breaks ◽

Telomere Maintenance ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Cellular Processes ◽

Molecular Functions ◽

Ku Proteins ◽

The Stability ◽

Dependent Processes

Ku proteins are associated with a variety of cellular processes such as repair of DNA-double-strand breaks, telomere maintenance and retrotransposition. In recent years, we have learned a lot about their cellular and molecular functions and it has turned out that Ku-dependent processes affect the stability of the genome, both positively and negatively, in several ways. This article gives an overview on the role of Ku in determining the shape of the genome.

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Mdt1 Facilitates Efficient Repair of Blocked DNA Double-Strand Breaks and Recombinational Maintenance of Telomeres

Molecular and Cellular Biology ◽

10.1128/mcb.00471-07 ◽

2007 ◽

Vol 27 (18) ◽

pp. 6532-6545 ◽

Cited By ~ 11

Author(s):

Brietta L. Pike ◽

Jörg Heierhorst

Keyword(s):

Dna Recombination ◽

Double Strand Breaks ◽

Telomere Maintenance ◽

Type I ◽

Dna Double Strand Breaks ◽

Drug Induced ◽

Exogenous Dna ◽

Strand Breaks ◽

Dramatic Shift ◽

Recombination Pathway

ABSTRACT DNA recombination plays critical roles in DNA repair and alternative telomere maintenance. Here we show that absence of the SQ/TQ cluster domain-containing protein Mdt1 (Ybl051c) renders Saccharomyces cerevisiae particularly hypersensitive to bleomycin, a drug that causes 3′-phospho-glycolate-blocked DNA double-strand breaks (DSBs). mdt1Δ also hypersensitizes partially recombination-defective cells to camptothecin-induced 3′-phospho-tyrosyl protein-blocked DSBs. Remarkably, whereas mdt1Δ cells are unable to restore broken chromosomes after bleomycin treatment, they efficiently repair “clean” endonuclease-generated DSBs. Epistasis analyses indicate that MDT1 acts in the repair of bleomycin-induced DSBs by regulating the efficiency of the homologous recombination pathway as well as telomere-related functions of the KU complex. Moreover, mdt1Δ leads to severe synthetic growth defects with a deletion of the recombination facilitator and telomere-positioning factor gene CTF18 already in the absence of exogenous DNA damage. Importantly, mdt1Δ causes a dramatic shift from the usually prevalent type II to the less-efficient type I pathway of recombinational telomere maintenance in the absence of telomerase in liquid senescence assays. As telomeres resemble protein-blocked DSBs, the results indicate that Mdt1 acts in a novel blocked-end-specific recombination pathway that is required for the efficiency of both drug-induced DSB repair and telomerase-independent telomere maintenance.

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Histone Modifications and the Maintenance of Telomere Integrity

Cells ◽

10.3390/cells8020199 ◽

2019 ◽

Vol 8 (2) ◽

pp. 199 ◽

Cited By ~ 8

Author(s):

Meagan Jezek ◽

Erin Green

Keyword(s):

Histone Modifications ◽

Current Knowledge ◽

Telomere Maintenance ◽

Model Systems ◽

Future Research ◽

Dna Double Strand Breaks ◽

Post Translational Modifications ◽

Strand Breaks ◽

Integral Role ◽

Protective Proteins

Telomeres, the nucleoprotein structures at the ends of eukaryotic chromosomes, play an integral role in protecting linear DNA from degradation. Dysregulation of telomeres can result in genomic instability and has been implicated in increased rates of cellular senescence and many diseases, including cancer. The integrity of telomeres is maintained by a coordinated network of proteins and RNAs, such as the telomerase holoenzyme and protective proteins that prevent the recognition of the telomere ends as a DNA double-strand breaks. The structure of chromatin at telomeres and within adjacent subtelomeres has been implicated in telomere maintenance pathways in model systems and humans. Specific post-translational modifications of histones, including methylation, acetylation, and ubiquitination, have been shown to be necessary for maintaining a chromatin environment that promotes telomere integrity. Here we review the current knowledge regarding the role of histone modifications in maintaining telomeric and subtelomeric chromatin, discuss the implications of histone modification marks as they relate to human disease, and highlight key areas for future research.

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Rad21 Haploinsufficiency Prevents ALT-Associated Phenotypes in Zebrafish Brain Tumors

Genes ◽

10.3390/genes11121442 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1442

Author(s):

Aurora Irene Idilli ◽

Cecilia Pazzi ◽

Francesca dal Pozzolo ◽

Michela Roccuzzo ◽

Maria Caterina Mione

Keyword(s):

Brain Tumors ◽

Telomere Maintenance ◽

Long Distance ◽

Zebrafish Model ◽

Strand Breaks ◽

Zebrafish Brain ◽

3D Genome ◽

Alternative Lengthening ◽

Repair Efficiency

Cohesin is a protein complex consisting of four core subunits responsible for sister chromatid cohesion in mitosis and meiosis, and for 3D genome organization and gene expression through the establishment of long distance interactions regulating transcriptional activity in the interphase. Both roles are important for telomere integrity, but the role of cohesin in telomere maintenance mechanisms in highly replicating cancer cells in vivo is poorly studied. Here we used a zebrafish model of brain tumor, which uses alternative lengthening of telomeres (ALT) as primary telomere maintenance mechanism to test whether haploinsufficiency for Rad21, a member of the cohesin ring, affects ALT development. We found that a reduction in Rad21 levels prevents ALT-associated phenotypes in zebrafish brain tumors and triggers an increase in tert expression. Despite the rescue of ALT phenotypes, tumor cells in rad21+/− fish exhibit an increase in DNA damage foci, probably due to a reduction in double-strand breaks repair efficiency.

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Mutant Telomeric Repeats in Yeast Can Disrupt the Negative Regulation of Recombination-Mediated Telomere Maintenance and Create an Alternative Lengthening of Telomeres-Like Phenotype

Molecular and Cellular Biology ◽

10.1128/mcb.00423-08 ◽

2008 ◽

Vol 29 (3) ◽

pp. 626-639 ◽

Cited By ~ 16

Author(s):

Laura H. Bechard ◽

Bilge D. Butuner ◽

George J. Peterson ◽

Will McRae ◽

Zeki Topcu ◽

...

Keyword(s):

Kluyveromyces Lactis ◽

Telomere Maintenance ◽

Telomeric Repeats ◽

Alternative Lengthening Of Telomeres ◽

Telomere Elongation ◽

Alternative Lengthening ◽

Telomere Capping ◽

Futile Cycles ◽

Telomeric Protein ◽

In Cells

ABSTRACT Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent of human ALT cells. In this study, we generated telomeres composed of either of two types of mutant telomeric repeats, Acc and SnaB, that each alter the binding site for the telomeric protein Rap1. We show here that arrays of both types of mutant repeats present basally on a telomere were defective in negatively regulating telomere length in the presence of telomerase. Similarly, when each type of mutant repeat was spread to all chromosome ends in cells lacking telomerase, they led to the formation of telomeres produced by RTE that were much longer than those seen in cells with only wild-type telomeric repeats. The Acc repeats produced the more severe defect in both types of telomere maintenance, consistent with their more severe Rap1 binding defect. Curiously, although telomerase deletion mutants with telomeres composed of Acc repeats invariably showed extreme telomere elongation, they often also initially showed persistent very short telomeres with few or no Acc repeats. We suggest that these result from futile cycles of recombinational elongation and truncation of the Acc repeats from the telomeres. The presence of extensive 3′ overhangs at mutant telomeres suggests that Rap1 may normally be involved in controlling 5′ end degradation.

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Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

Journal of Histochemistry & Cytochemistry ◽

10.1369/0022155418761351 ◽

2018 ◽

Vol 66 (7) ◽

pp. 485-495 ◽

Cited By ~ 2

Author(s):

Aernoud A. van Batenburg ◽

Karin M. Kazemier ◽

Ton Peeters ◽

Matthijs F. M. van Oosterhout ◽

Joanne J. van der Vis ◽

...

Keyword(s):

Smooth Muscle ◽

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Telomere Length ◽

Dna Sequences ◽

Double Strand Breaks ◽

Telomere Maintenance ◽

Dna Double Strand Breaks ◽

Strand Breaks

Telomeres are small repetitive DNA sequences at the ends of chromosomes which act as a buffer in age-dependent DNA shortening. Insufficient telomere repeats will be recognized as double-strand breaks. Presently, it is becoming more evident that telomere attrition, whether or not caused by mutations in telomere maintenance genes, plays an important role in many inflammatory and age-associated diseases. In this report, a method to (semi)quantitatively assess telomere length and DNA double-strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue is described. Therefore, a novel combination of quantitative fluorescence in situ hybridization, tissue elution, and immunofluorescence staining techniques was developed. Caveolin-1 (type 1 pneumocytes), pro-surfactant protein C (type 2 pneumocytes), club cell-10 (club cells), and alpha smooth muscle actin (smooth muscle cells) markers were used to identify cell types. To visualize all the different probes, restaining the tissue by heat-mediated slide elution is essential. Fluorescent signals of telomeres and DNA double-strand breaks were quantified using the Telometer plugin of ImageJ. As example, we analyzed lung tissue from a familial pulmonary fibrosis patient with a mutation in the telomere-associated gene poly(A)-specific ribonuclease ( PARN). The protocol displays a novel opportunity to directly quantitatively link DNA double-strand breaks to telomere length in specific FFPE cells.

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CBIO-08. ENDOGENOUS DNA DOUBLE STRAND BREAKS ACTIVATE HETEROGENOUS DNA DAMAGE SIGNALING IN IDH1/2 MUTANT GLIOMAS

Neuro-Oncology ◽

10.1093/neuonc/noab196.109 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi28-vi29

Author(s):

Gaspar Kitange ◽

Rachael Vaubel ◽

Jann Sarkaria

Keyword(s):

Dna Damage ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Isocitrate Dehydrogenase 1 ◽

Strand Breaks ◽

Dna Damage Signaling ◽

Alternative Lengthening ◽

Systematic Effort ◽

Patient Specimen

Abstract Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.

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Alternative Lengthening of Telomeres: Lessons to Be Learned from Telomeric DNA Double-Strand Break Repair

Genes ◽

10.3390/genes12111734 ◽

2021 ◽

Vol 12 (11) ◽

pp. 1734

Author(s):

Thomas Kent ◽

David Clynes

Keyword(s):

Strand Break ◽

Dna Double Strand Breaks ◽

Telomeric Dna ◽

Dsb Repair ◽

Alternative Lengthening Of Telomeres ◽

Strand Breaks ◽

Alternative Lengthening ◽

Dna Double Strand Break ◽

Cycle Dependent ◽

Telomere Lengthening

The study of the molecular pathways underlying cancer has given us important insights into how breaks in our DNA are repaired and the dire consequences that can occur when these processes are perturbed. Extensive research over the past 20 years has shown that the key molecular event underpinning a subset of cancers involves the deregulated repair of DNA double-strand breaks (DSBs) at telomeres, which in turn leads to telomere lengthening and the potential for replicative immortality. Here we discuss, in-depth, recent major breakthroughs in our understanding of the mechanisms underpinning this pathway known as the alternative lengthening of telomeres (ALT). We explore how this gives us important insights into how DSB repair at telomeres is regulated, with relevance to the cell-cycle-dependent regulation of repair, repair of stalled replication forks and the spatial regulation of DSB repair.

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H2AX regulates meiotic telomere clustering

The Journal of Cell Biology ◽

10.1083/jcb.200305124 ◽

2003 ◽

Vol 163 (1) ◽

pp. 15-20 ◽

Cited By ~ 38

Author(s):

Oscar Fernandez-Capetillo ◽

Bodo Liebe ◽

Harry Scherthan ◽

André Nussenzweig

Keyword(s):

Ataxia Telangiectasia ◽

Essential Role ◽

Double Strand Breaks ◽

Telomere Maintenance ◽

Histone H2a ◽

Dna Double Strand Breaks ◽

Ataxia Telangiectasia Mutated ◽

Strand Breaks ◽

Downstream Effector ◽

Chromosome Fusions

The histone H2A variant H2AX is phosphorylated in response to DNA double-strand breaks originating from diverse origins, including dysfunctional telomeres. Here, we show that normal mitotic telomere maintenance does not require H2AX. Moreover, H2AX is dispensable for the chromosome fusions arising from either critically shortened or deprotected telomeres. However, H2AX has an essential role in controlling the proper topological distribution of telomeres during meiotic prophase I. Our results suggest that H2AX is a downstream effector of the ataxia telangiectasia–mutated kinase in controlling telomere movement during meiosis.

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