scholarly journals Nucleostemin prevents telomere damage by promoting PML-IV recruitment to SUMOylated TRF1

2012 ◽  
Vol 197 (5) ◽  
pp. 613-624 ◽  
Author(s):  
Joseph K. Hsu ◽  
Tao Lin ◽  
Robert Y.L. Tsai
Keyword(s):  
Dna Damage ◽  
Proliferative Potential ◽  
Telomere Protection ◽  
Alternative Lengthening ◽  
Pml Bodies ◽  
Dividing Cells ◽  
Telomeric Length

Continuously dividing cells must be protected from telomeric and nontelomeric DNA damage in order to maintain their proliferative potential. Here, we report a novel telomere-protecting mechanism regulated by nucleostemin (NS). NS depletion increased the number of telomere damage foci in both telomerase-active (TA+) and alternative lengthening of telomere (ALT) cells and decreased the percentage of damaged telomeres associated with ALT-associated PML bodies (APB) and the number of APB in ALT cells. Mechanistically, NS could promote the recruitment of PML-IV to SUMOylated TRF1 in TA+ and ALT cells. This event was stimulated by DNA damage. Supporting the importance of NS and PML-IV in telomere protection, we demonstrate that loss of NS or PML-IV increased the frequency of telomere damage and aberration, reduced telomeric length, and perturbed the TRF2ΔBΔM-induced telomeric recruitment of RAD51. Conversely, overexpression of either NS or PML-IV protected ALT and TA+ cells from telomere damage. This work reveals a novel mechanism in telomere protection.

Sumoylation of TRF1 Is Essential for Its Recruitment to ALT-Associated PML Bodies.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4169-4169
Author(s):  
Jian Yu ◽  
Jianping Lan ◽  
Yuanyuan Zhu ◽  
Xiaoyu Lai ◽  
He Huang
Keyword(s):  
Cancer Cells ◽  
Computational Prediction ◽  
Proliferative Potential ◽  
Telomeric Dna ◽  
Alternative Lengthening ◽  
Pml Bodies ◽  
U2os Cells ◽  
Telomeric Binding Protein

Abstract To achieve unlimited proliferative potential, most cancer cells activate telomerase to maintain telomeres. However, some cancer cells elongate telomeres through a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). These ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB), which comprises telomeric DNA and a number of proteins, including PML protein, the telomere binding proteins TRF1 and TRF2, replication factor A, and recombination factors Rad51, Rad52, and the Rad50/Mre11/NBS1 complex. TRF1, as the first identified telomeric binding protein, binds the duplex telomeric repeats at telomere ends. It plays an important role in telomere length control, telomeric ends shelter and cell cycle regulation. In ALT cell lines, TRF1 co-localized with PML protein at APBs, but the exact mechanism of its recruitment to APBs is not clear. Here we show that TRF1 localizes to PML bodies in about 5% of an asynchronously growing culture of U2OS cells and the percentage of cells with colocalization of TRF1 and PML bodies increases to about 40% in cells enriched in G2/M, which is consistent with the previous studies. Furthermore, our results show that TRF1 is modified by the small ubiquitin-like protein SUMO-1 in vivo and in vitro. Firstly, 293T cells were transfected with Flag-TRF1, HA-Ubc9, GFP-SUMO1 and then immunoprecipitated by using FLAG-M2 gel under denaturing conditions. Immunoblotting with GFP and Flag antibodies demonstrated that TRF1 is modified by SUMO-1 in vivo. Next, in vitro SUMO-1 conjugation assay of TRF1 was employed. The results showed that TRF1 is conjugated with SUMO-1 in the presence of purified recombinant protein SAE1/SAE2, Ubc9, SUMO1, His-TRF1 and ATP. Either SAE1/SAE2, Ubc9, SUMO-1 or ATP was omitted from the reaction abolished the sumoylation of TRF1. Previous studies have shown that sumoylation controls the recruitment of several proteins to PML bodies, so we examined whether sumoylation of TRF1 is required for its recruitment to APBs. We mutated the potential sumoylation sites of TRF1 according to the computational prediction and then transfected it into U2OS cells to examine its localization. The results showed that TRF1 mutant does not localize to PML bodies. Taken together, all these results suggest that TRF is modified by SUMO-1 and sumoylation of TRF1 is essential for its recruitment to APBs in ALT cells.

scholarly journals FANCM suppresses DNA replication stress at ALT telomeres by disrupting TERRA R-loops

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xiaolei Pan ◽  
Yun Chen ◽  
Beena Biju ◽  
Naveed Ahmed ◽  
Joyce Kong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Replication Stress ◽  
Checkpoint Kinases ◽  
Alternative Lengthening ◽  
Pml Bodies ◽  
Damage Response ◽  
Dna Replication Stress ◽  
The Many ◽  
Break Induced Replication

AbstractCancer cells maintain their telomeres by either re-activating telomerase or adopting the homologous recombination (HR)-based Alternative Lengthening of Telomere (ALT) pathway. Among the many prominent features of ALT cells, C-circles (CC) formation is considered to be the most specific and quantifiable biomarker of ALT. However, the molecular mechanism behind the initiation and maintenance of CC formation in ALT cells is still largely unknown. We reported previously that depletion of the FANCM complex (FANCM-FAAP24-MHF1&2) in ALT cells induced pronounced replication stress, which primarily takes place at their telomeres. Here, we characterized the changes in ALT associated phenotypes in cells deficient of the FANCM complex. We found that depletion of FAAP24 or FANCM, but not MHF1&2, induces a dramatic increase of CC formation. Most importantly, we identified multiple DNA damage response (DDR) and DNA repair pathways that stimulate the dramatic increase of CC formation in FANCM deficient cells, including the dissolvase complex (BLM-TOP3A-RMI1/2, or BTR), DNA damage checkpoint kinases (ATR and Chk1), HR proteins (BRCA2, PALB2, and Rad51), as well as proteins involved in Break-Induced Replication (BIR) (POLD1 and POLD3). In addition, FANCD2, another Fanconi Anemia (FA) protein, is also required for CC formation, likely through promoting the recruitment of BLM to the replication stressed ALT telomeres. Finally, we demonstrated that TERRA R-loops accumulate at telomeres in FANCM deficient ALT cells and downregulation of which attenuates the ALT-associated PML bodies (APBs), replication stress and CC formation. Taken together, our data suggest that FANCM prevents replisomes from stalling/collapsing at ALT telomeres by disrupting TERRA R-loops.

scholarly journals Bmi1 Augments Proliferation and Survival of Cortical Bone-Derived Stem Cells after Injury through Novel Epigenetic Signaling via Histone 3 Regulation

2021 ◽  
Vol 22 (15) ◽  
pp. 7813
Author(s):  
Lindsay Kraus ◽  
Chris Bryan ◽  
Marcus Wagner ◽  
Tabito Kino ◽  
Melissa Gunchenko ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Dna Damage ◽  
Stem Cell ◽  
Histone Modification ◽  
Epigenetic Regulation ◽  
Critical Role ◽  
Proliferative Potential ◽  
Therapeutic Effects ◽  
Histone 3

Ischemic heart disease can lead to myocardial infarction (MI), a major cause of morbidity and mortality worldwide. Multiple stem cell types have been safely transferred into failing human hearts, but the overall clinical cardiovascular benefits have been modest. Therefore, there is a dire need to understand the basic biology of stem cells to enhance therapeutic effects. Bmi1 is part of the polycomb repressive complex 1 (PRC1) that is involved in different processes including proliferation, survival and differentiation of stem cells. We isolated cortical bones stem cells (CBSCs) from bone stroma, and they express significantly high levels of Bmi1 compared to mesenchymal stem cells (MSCs) and cardiac-derived stem cells (CDCs). Using lentiviral transduction, Bmi1 was knocked down in the CBSCs to determine the effect of loss of Bmi1 on proliferation and survival potential with or without Bmi1 in CBSCs. Our data show that with the loss of Bmi1, there is a decrease in CBSC ability to proliferate and survive during stress. This loss of functionality is attributed to changes in histone modification, specifically histone 3 lysine 27 (H3K27). Without the proper epigenetic regulation, due to the loss of the polycomb protein in CBSCs, there is a significant decrease in cell cycle proteins, including Cyclin B, E2F, and WEE as well as an increase in DNA damage genes, including ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). In conclusion, in the absence of Bmi1, CBSCs lose their proliferative potential, have increased DNA damage and apoptosis, and more cell cycle arrest due to changes in epigenetic modifications. Consequently, Bmi1 plays a critical role in stem cell proliferation and survival through cell cycle regulation, specifically in the CBSCs. This regulation is associated with the histone modification and regulation of Bmi1, therefore indicating a novel mechanism of Bmi1 and the epigenetic regulation of stem cells.

scholarly journals G-quadruplex Stabilization Fuels the ALT Pathway in ALT-positive Osteosarcoma Cells

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 304 ◽  
Author(s):  
Roberta Amato ◽  
Martina Valenzuela ◽  
Francesco Berardinelli ◽  
Erica Salvati ◽  
Carmen Maresca ◽  
...  
Keyword(s):  
Dna Damage ◽  
Sister Chromatid Exchanges ◽  
Telomere Maintenance ◽  
Osteosarcoma Cell ◽  
Telomeric Dna ◽  
Replicative Stress ◽  
Alternative Lengthening ◽  
G Quadruplex ◽  
Reduce Cell Proliferation ◽  
The Impact

Most human tumors maintain telomere lengths by telomerase, whereas a portion of them (10–15%) uses a mechanism named alternative lengthening of telomeres (ALT). The telomeric G-quadruplex (G4) ligand RHPS4 is known for its potent antiproliferative effect, as shown in telomerase-positive cancer models. Moreover, RHPS4 is also able to reduce cell proliferation in ALT cells, although the influence of G4 stabilization on the ALT mechanism has so far been poorly investigated. Here we show that sensitivity to RHPS4 is comparable in ALT-positive (U2OS; SAOS-2) and telomerase-positive (HOS) osteosarcoma cell lines, unlinking the telomere maintenance mechanism and RHPS4 responsiveness. To investigate the impact of G4 stabilization on ALT, the cardinal ALT hallmarks were analyzed. A significant induction of telomeric doublets, telomeric clusterized DNA damage, ALT-associated Promyelocytic Leukaemia-bodies (APBs), telomere sister chromatid exchanges (T-SCE) and c-circles was found exclusively in RHPS4-treated ALT cells. We surmise that RHPS4 affects ALT mechanisms through the induction of replicative stress that in turn is converted in DNA damage at telomeres, fueling recombination. In conclusion, our work indicates that RHPS4-induced telomeric DNA damage promotes overactivation of telomeric recombination in ALT cells, opening new questions on the therapeutic employment of G4 ligands in the treatment of ALT positive tumors.

Imatinib Increases Cytotoxicity of Melphalan and Their Combination Allows An Efficient Killing of CML Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4254-4254
Author(s):  
Cesarina Giallongo ◽  
Piera La Cava ◽  
Daniele Tibullo ◽  
Nunziatina Parrinello ◽  
Provvidenza Guagliardo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Drug Resistance ◽  
Dna Repair ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Proliferative Potential ◽  
Cell Accumulation ◽  
M Phase ◽  
Pre Treatment ◽  
Human Leukemic Cells

Abstract Abstract 4254 BCR/ABL-positive cells are relatively resistant to chemotherapy and, in order to evaluate the effect of Imatinib (IM) in reverting drug-resistance, we evaluated on K562 the toxicity of 1 h exposure to cytosine arabinoside (ARA-C) 20 μM, hydroxyurea (HU) 100 μM, and melphalan (MEL) 20 μM, after a pre-treatment of 24 h with 1 μM IM. The doses of the drugs were similar to that achieved in the plasma after standard chemoterapeutic treatment. Cell viability was evaluated by ATP-lite at 24, 48 and 72 hs from beginning of drug-free condition. The combinations of IM plus MEL induced the highest cytotoxicity (P<0,001 at 24, 48 and 72 hs vs MEL alone) indicating that pre-treatment with IM increased K562 exposition to the genotoxic damage of MEL. We next analyzed effects on cell cycle and DNA damage by alkaline comet assay induced by this drug combination and we observed that DNA damage peaked at 48 h with IM/MEL combination. In addition, flow cytometry analysis showed that IM/MEL combination reduced the cell accumulation in G2/M phase induced by MEL (P<0.001 vs MEL), thus reducing the ability to DNA repair and recovery. These data indicate that inhibition of BCR/ABL activity by IM increased cell cytotoxicity of MEL by reducing the effectiveness of the DNA-repair pathways and decreasing the time for DNA repair at the G2/M checkpoint.. To ascertain that these results were linked to BCR/ABL inhibition, TonB.210, a cell line where the BCR-ABL expression is inducible by doxycycline (DOX), were treated in the same conditions. Only TonB.210 cultured with DOX were insensitive to MEL while IM/MEL combination reverted these drug resistance (P<0,001). In the final step we studied the sequential association IM/MEL on the proliferative potential of myeloid progenitors of 6 CML patients at diagnosis. IM/MEL combination increased the reduction of the overall number of colonies in comparison to IM alone (P<0.05 vs IM). In addition, the analysis on CFU-GM and BFU-E colonies by qRT-PCR demonstrated that the IM/MEL combination led to the highest reduction in the number of BCR/ABL positive colonies (P<0.01 vs IM). Therefore, our data indicate that the pre-inhibition of BCR/ABL activity by IM increases the toxicity of MEL and allows an efficient killing of human leukemic cells, thus suggesting new therapeutic combinations for CML patients Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evidence for alternative lengthening of telomeres in liposarcomas in the absence of ALT-associated PML bodies

2008 ◽  
Vol 122 (11) ◽  
pp. 2414-2421 ◽  
Author(s):  
Jennie N. Jeyapalan ◽  
Aaron Mendez-Bermudez ◽  
Nadia Zaffaroni ◽  
Yuri E. Dubrova ◽  
Nicola J. Royle
Keyword(s):  
Alternative Lengthening Of Telomeres ◽  
Alternative Lengthening ◽  
Pml Bodies

scholarly journals Werner Protein Protects Nonproliferating Cells from Oxidative DNA Damage

2005 ◽  
Vol 25 (23) ◽  
pp. 10492-10506 ◽  
Author(s):  
Anna M. Szekely ◽  
Franziska Bleichert ◽  
Astrid Nümann ◽  
Stephen Van Komen ◽  
Elisabeth Manasanch ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Oxidative Dna Damage ◽  
Werner Syndrome ◽  
Human Fibroblasts ◽  
Telomere Maintenance ◽  
Telomere Binding Protein ◽  
Damage Response ◽  
Dividing Cells

ABSTRACT Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of γH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.

scholarly journals Epigenetic Telomere Protection by Drosophila DNA Damage Response Pathways

PLoS Genetics ◽  
2006 ◽  
Vol 2 (5) ◽  
pp. e71 ◽  
Author(s):  
Sarah R Oikemus ◽  
Joana Queiroz-Machado ◽  
KuanJu Lai ◽  
Nadine McGinnis ◽  
Claudio Sunkel ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Telomere Protection ◽  
Damage Response

scholarly journals Role of PROP1 in Pituitary Gland Growth

2005 ◽  
Vol 19 (3) ◽  
pp. 698-710 ◽  
Author(s):  
Robert D. Ward ◽  
Lori T. Raetzman ◽  
Hoonkyo Suh ◽  
Brandon M. Stone ◽  
Igor O. Nasonkin ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Anterior Pituitary ◽  
Anterior Lobe ◽  
Clinical Findings ◽  
Proliferative Potential ◽  
Rathke’S Pouch ◽  
Rathke's Pouch ◽  
Pituitary Hyperplasia ◽  
Dividing Cells ◽  
Pituitary Hypoplasia

Abstract Mutations in the PROP1 transcription factor gene lead to reduced production of thyrotropin, GH, prolactin, and gonadotropins as well as to pituitary hypoplasia in adult humans and mice. Some PROP1-deficient patients initially exhibit pituitary hyperplasia that resolves to hypoplasia. To understand this feature and to explore the mechanism whereby PROP1 regulates anterior pituitary gland growth, we carried out longitudinal studies in normal and Prop1-deficient dwarf mice from early embryogenesis through adulthood, examining the volume of Rathke’s pouch and its derivatives, the position and number of dividing cells, the rate of apoptosis, and cell migration by pulse labeling. The results suggest that anterior pituitary progenitors normally leave the perilumenal region of Rathke’s pouch and migrate to form the anterior lobe as they differentiate. Some of the cells that seed the anterior lobe during organogenesis have proliferative potential, supporting the expansion of the anterior lobe after birth. Prop1-deficient fetal pituitaries are dysmorphic because mutant cells are retained in the perilumenal area and fail to differentiate. After birth, mutant pituitaries exhibit enhanced apoptosis and reduced proliferation, apparently because the mutant anterior lobe is not seeded with progenitors. These studies suggest a mechanism for Prop1 action and an explanation for some of the clinical findings in human patients.

scholarly journals RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage–induced cell senescence

2016 ◽  
Vol 27 (8) ◽  
pp. 1346-1357 ◽  
Author(s):  
Pavol Cekan ◽  
Keisuke Hasegawa ◽  
Yu Pan ◽  
Emily Tubman ◽  
David Odde ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Progression ◽  
Nucleotide Exchange ◽  
Normal Cells ◽  
Cytoplasmic Transport ◽  
Dividing Cells ◽  
Ran Gtp

The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase–regulated nuclear–cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage–induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β–dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP–regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage.

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