The Impact of Telomere Shortening in Dyskeratosis Congenita Cells on DNA Damage Response Pathways.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4052-4052
Author(s):  
Travis Witt ◽  
Aloysius Klingelhutz ◽  
Erik Westin ◽  
Preeti Satyanarayana ◽  
Peter M. Lansdorp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Cellular Proliferation ◽  
Bone Marrow Failure ◽  
Telomerase Activity ◽  
Dyskeratosis Congenita ◽  
Cytotoxic Agents ◽  
Damage Response ◽  
And Control

Abstract Dyskeratosis congenita (DC) is an inherited multisystem disorder of premature aging, typically characterized by bone marrow failure, mucosal leukoplakia, abnormal skin pigmentation, and nail dystrophy. The X-linked and autosomal dominant forms of DC are associated with mutations in genes that affect telomerase activity resulting in a decrease in telomere length. DC, like other bone marrow failure disorders, is associated with ineffective hematopoiesis and a cancer predisposition. Standard treatment of bone marrow failure or cancer requires cytotoxic therapy, and clinical observations suggest DC patients have an increased sensitivity to cytotoxic therapy. To explain this, we hypothesized that the short telomeres in somatic cells from DC patients could alter the activity and/or expression of several proteins involved in DNA repair or the response to cellular stress including p16, p53 and p21. Lymphocytes from five DC subjects and age-matched controls were stimulated to grow in vitro in the presence of various cytotoxic agents with different modes of action, including Taxol (antimitotic agent and microtubule inhibitor) and Etoposide (topoisomerase inhibitor and DNA damaging agent). In addition, we tested fibroblasts and keratinocyte extracted from skin biopsies from DC and control subjects that were serially passaged. Cellular proliferation and cell death were monitored by cell counts and flow cytometry. Western blotting was used to measure steady state and DNA damage- induced expression of tumor suppressor protein p53 and other proteins involved in DNA damage response signaling pathway, including p16 and p21 in relation to telomere length. Results of flow cytometry accompanied by direct visualization showed a decreased proliferation of DC lymphocytes compared to normal cells, and this growth disadvantage was further accentuated following cell exposure to cytotoxic agents. DC lymphocytes exposed to 10−6 M Taxol showed a decrease in cellular proliferation between 3 and 8 fold while normal control cells exposed to the same agents exhibited only a 3 to 4 fold decrease in cell growth. Similarly DC lymphocytes exposed to Etoposide were inhibited to a greater extent than control cells. Western blot analysis of whole cell lysates indicated a difference in DNA damage response proteins. Of note, lymphocytes from several DC subjects exposed to Taxol did not upregulate p53 expression, while inducible levels were noted in Taxol-treated control cells. In contrast, DC and control lymphocytes exposed to Etoposide upregulated p53 in a similar dose dependent manner. No differences were noted in DC versus control lymphocytes with regards to basal or chemotherapy induced p16 expression. Interestingly, late passage DC fibroblasts displayed enhanced basal expression of p16. These results support the clinical observation of increased “chemosensitivity” in DC subjects and suggest that diminished telomerase activity and premature telomere shortening may interfere with normal DNA damage and stress response pathways. These data are also consistent with our finding that DC fibroblasts, keratinocytes, and lymphocytes have a reduced cell proliferative lifespan. Further studies are needed to dissect the role of telomeres in the cellular response to various types of DNA damage.

scholarly journals Pot1b Deletion and Telomerase Haploinsufficiency in Mice Initiate an ATR-Dependent DNA Damage Response and Elicit Phenotypes Resembling Dyskeratosis Congenita

2008 ◽  
Vol 29 (1) ◽  
pp. 229-240 ◽  
Author(s):  
Hua He ◽  
Yang Wang ◽  
Xiaolan Guo ◽  
Sonal Ramchandani ◽  
Jin Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Bone Marrow Failure ◽  
Chromosome Instability ◽  
Premature Death ◽  
Dyskeratosis Congenita ◽  
Damage Response ◽  
Survival Potential

ABSTRACT The Protection of telomeres 1 (POT1) protein is a single-stranded telomere binding protein that is essential for proper maintenance of telomere length. Disruption of POT1 function leads to chromosome instability and loss of cellular viability. Here, we show that targeted deletion of the mouse Pot1b gene results in increased apoptosis in highly proliferative tissues. In the setting of telomerase haploinsufficiency, loss of Pot1b results in depletion of germ cells and complete bone marrow failure due to increased apoptosis, culminating in premature death. Pot1b −/ − mTR +/ − hematopoietic progenitor and stem cells display markedly reduced survival potential in vitro. Accelerated telomere shortening, increased G overhang and elevated number of chromosome end-to-end fusions that initiate an ATR-dependent DNA damage response were also observed. These results indicate an essential role for Pot1b in the maintenance of genome integrity and the long-term viability of proliferative tissues in the setting of telomerase deficiency. Interestingly, these phenotypes closely resemble those found in the human disease dyskeratosis congenita (DC), an inherited syndrome characterized by bone marrow failure, hyperpigmentation, and nail dystrophy. We anticipate that this mouse will serve as a useful model to further understand the pathophysiology of DC.

HSC Exit From Dormancy Provokes De Novo DNA Damage, Leading To Bone Marrow Failure If Unresolved By The Fanconi Anemia Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 799-799
Author(s):  
Dagmar Walter ◽  
Amelie Lier ◽  
Anja Geiselhart ◽  
Sina Huntscha ◽  
David Brocks ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Aplastic Anemia ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
De Novo ◽  
Bone Marrow Failure ◽  
Damage Response ◽  
Fold Induction

Abstract Long-term quiescence has been proposed to preserve the genomic stability of hematopoietic stem cells (HSCs) during aging. The current models of HSC aging are limited in their ability to observe both DNA damage in vivo and the consequences of this damage upon hematopoiesis. Fanconi Anemia (FA) is a hereditary multisystem disorder, characterized by defective DNA damage response and progressive bone marrow failure in most patients. However, the existing genetic models of FA do not develop aplastic anemia, suggesting that cell-extrinsic factors may play a causal role. We sought to identify whether physiologic mediators of HSC activation could be used as agonists to provoke DNA damage and HSC attrition in vivo. Mice were treated with a range of agonists that promote the in vivo exit of HSC from a dormant state into active cycling (polyI:polyC; Interferon-α; G-CSF; TPO; and serial bleeding). Highly purified HSC demonstrated a rapid 3-5-fold induction of DNA damage after treatment with all agonists (p<0.01), as assessed by both enumerating γ-H2AX foci and by alkaline comet assay. Mechanistically, stress-induced exit from quiescence correlated with increased mitochondrial metabolism in HSC, as evaluated by elevated mitochondrial membrane potential (2-fold increased, p<0.01) and superoxide levels (1.5-fold increased, p<0.05). Critically, we could directly implicate these reactive oxygen species in DNA damage as we observed a 1.4-fold increase in 8-Oxo-dG lesions in HSC that had been activated into cycle in vivo(p<0.05). At 48 h post-treatment, γ-H2AX levels began to decrease and this repair was concomitant with an induction of the FA signaling pathway in HSC, as demonstrated by both increased levels of FA gene expression and elevated FANCD2 foci (4-fold induction, p<0.01). Treatment of Fanca-/- mice with polyI:polyC led to a HSC proliferative response comparable to wild type (WT) mice but resulted in a 2-fold higher level of activation-induced DNA damage (p<0.05), demonstrating that this repair pathway is involved in resolving activation-induced DNA damage. Four rounds of serial in vivo activation led to a permanent depletion of the most primitive label-retaining Fanca-/- HSC and this correlated with a 4-fold depletion of functional HSC (p<0.01) as defined by competitive repopulation assays. Subsequent rounds of HSC activation with polyI:polyC resulted in the onset of a severe aplastic anemia (SAA) in 33% of treated Fanca-/- mice but not in any of the WT controls. SSA was characterized by a dramatic reduction in bone marrow (BM) cellularity, profound thrombocytopenia (21-246x106 platelets/ml), leukocytopenia (0.4-0.5x106 WBC/ml), neutropenia (0.03-0.1x106/ml) and anemia (1.5-2.3 g/dL Hb). Examination of BM HSC/progenitors demonstrated nearly complete loss of HSC, MPP, CMP and CLP (depletion of ≥33x, 8x, 4x and 12x respectively compared to PBS-treated Fanca-/-controls). Taken together, these data demonstrates that enforced exit from dormancy in vivo leads to de novo DNA damage in HSC, which is repaired by activation of a FA-dependent DNA damage response. Furthermore, the highly penetrant bone marrow failure observed in Fanconi anemia patients can be recapitulated by the serial application of a physiologic HSC activating signal to Fanca-/- mice. This suggests that the BM failure in FA may be caused by an aberrant response to HSC activation, most likely during exposure to infection or other physiologic stressors. These data provides a novel link between pro-inflammatory cytokines, DNA damage and HSC dysfunction and may have important clinical implications relevant to both prevention of BM failure in FA and in the study of age-related hematopoietic defects in non-FA patients. Moreover, these data provide the first evidence that FA knockout mouse models accurately recapitulate and provide novel insights into the etiology of BM failure in patients with FA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Monitoring and treatment of MDS in genetically susceptible persons

Hematology ◽  
2019 ◽  
Vol 2019 (1) ◽  
pp. 105-109 ◽  
Author(s):  
Stella M. Davies
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Bone Marrow Failure ◽  
Treatment Strategies ◽  
Informed Choice ◽  
Dyskeratosis Congenita ◽  
Careful Consideration ◽  
Host Susceptibility ◽  
Bone Marrow Failure Syndromes ◽  
Severe Infections

Abstract Genetic susceptibility to myelodysplastic syndrome (MDS) occurs in children with inherited bone marrow failure syndromes, including Fanconi anemia, Shwachman Diamond syndrome, and dyskeratosis congenita. Available evidence (although not perfect) supports annual surveillance of the blood count and bone marrow in affected persons. Optimal treatment of MDS in these persons is most commonly transplantation. Careful consideration must be given to host susceptibility to DNA damage when selecting a transplant strategy, because significant dose reductions and avoidance of radiation are necessary. Transplantation before evolution to acute myeloid leukemia (AML) is optimal, because outcomes of AML are extremely poor. Children and adults can present with germline mutations in GATA2 and RUNX1, both of which are associated with a 30% to 40% chance of evolution to MDS. GATA2 deficiency may be associated with a clinically important degree of immune suppression, which can cause severe infections that can complicate transplant strategies. GATA2 and RUNX1 deficiency is not associated with host susceptibility to DNA damage, and therefore, conventional treatment strategies for MDS and AML can be used. RUNX1 deficiency has a highly variable phenotype, and MDS can occur in childhood and later in adulthood within the same families, making annual surveillance with marrow examination burdensome; however, such strategies should be discussed with affected persons, allowing an informed choice.

scholarly journals PIMREG expression level predicts glioblastoma patient survival and affects temozolomide resistance and DNA damage response

2021 ◽  
Author(s):  
Rodolfo Bortolozo Serafim ◽  
Cibele Cardoso ◽  
Vanessa Arfelli ◽  
Valeria Valente ◽  
Leticia Fröhlich Archangelo
Keyword(s):  
Dna Damage ◽  
Nervous System ◽  
Dna Damage Response ◽  
Cellular Proliferation ◽  
Patient Survival ◽  
Glioblastoma Cells ◽  
Damage Response ◽  
Genotoxic Agents ◽  
And Migration ◽  
And Function

Abstract PIMREG expression strongly correlates with cellular proliferation in both malignant and normal cells. Throughout embryo development, PIMREG expression is prominent at the central nervous system. Recent studies have described high levels of PIMREG transcripts in different types of tumors and correlated with patient survival and tumor aggressiveness. Given the emerging significance of PIMREG in carcinogenesis and its putative role in the context of the nervous system, we investigated the expression and function of PIMREG in gliomas, the most common primary brain tumors. We performed an extensive analysis of PIMREG expression in tumors samples of glioma patients, assessed the effects of PIMREG silencing and overexpression on the sensitivity of glioblastoma cell lines treated with genotoxic agents commonly used for treating patients and assessed for treatment response, proliferation and migration. We show that glioblastoma exhibits the highest levels of PIMREG expression among all cancers analyzed and that elevated PIMREG expression is a biomarker for glioma progression and patient outcome. Moreover, PIMREG is induced by genotoxic agents and its silencing renders glioblastoma cells sensitive to temozolomide treatment and affects ATR- and ATM-dependent signaling. Our data demonstrate that PIMREG plays a role in DNA damage response and temozolomide resistance of glioblastoma cells and further support the PIMREG role in tumorigenesis.

Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 723-723
Author(s):  
Alexandra Sobeck ◽  
Stacie Stone ◽  
Bendert deGraaf ◽  
Vincenzo Costanzo ◽  
Johan deWinter ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
S Phase ◽  
Dependent Manner ◽  
Core Complex ◽  
Damage Response ◽  
Crosslinking Agents

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

scholarly journals Conditional TRF1 knockout in the hematopoietic compartment leads to bone marrow failure and recapitulates clinical features of dyskeratosis congenita

Blood ◽  
2012 ◽  
Vol 120 (15) ◽  
pp. 2990-3000 ◽  
Author(s):  
Fabian Beier ◽  
Miguel Foronda ◽  
Paula Martinez ◽  
Maria A. Blasco
Keyword(s):  
Bone Marrow ◽  
Clinical Features ◽  
Bone Marrow Failure ◽  
Telomerase Activity ◽  
Dyskeratosis Congenita ◽  
Telomere Shortening ◽  
Interacting Protein ◽  
Compensatory Proliferation ◽  
Allelic Variations

Abstract TRF1 is part of the shelterin complex, which binds telomeres and it is essential for their protection. Ablation of TRF1 induces sister telomere fusions and aberrant numbers of telomeric signals associated with telomere fragility. Dyskeratosis congenita is characterized by a mucocutaneous triad, bone marrow failure (BMF), and presence of short telomeres because of mutations in telomerase. A subset of patients, however, show mutations in the shelterin component TIN2, a TRF1-interacting protein, presenting a more severe phenotype and presence of very short telomeres despite normal telomerase activity. Allelic variations in TRF1 have been found associated with BMF. To address a possible role for TRF1 dysfunction in BMF, here we generated a mouse model with conditional TRF1 deletion in the hematopoietic system. Chronic TRF1 deletion results in increased DNA damage and cellular senescence, but not increased apoptosis, in BM progenitor cells, leading to severe aplasia. Importantly, increased compensatory proliferation of BM stem cells is associated with rapid telomere shortening and further increase in senescent cells in vivo, providing a mechanism for the very short telomeres of human patients with mutations in the shelterin TIN2. Together, these results represent proof of principle that mutations in TRF1 lead to the main clinical features of BMF.

scholarly journals Biomarkers of DNA Damage Response Enable Flow Cytometry-Based Diagnostic to Identify Inborn DNA Repair Defects in Primary Immunodeficiencies

2021 ◽  
Author(s):  
Kerstin Felgentreff ◽  
Ulrich Baumann ◽  
Christian Klemann ◽  
Catharina Schuetz ◽  
Dorothee Viemann ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Cancer Susceptibility ◽  
Bone Marrow Failure ◽  
Dna Lesions ◽  
Diagnostic Tools ◽  
Cellular Models ◽  
Damage Response

AbstractDNA damage is a constant event in every cell caused by exogenous factors such as ultraviolet and ionizing radiation (UVR/IR) and intercalating drugs, or endogenous metabolic and replicative stress. Proteins of the DNA damage response (DDR) network sense DNA lesions and induce cell cycle arrest, DNA repair, and apoptosis. Genetic defects of DDR or DNA repair proteins can be associated with immunodeficiency, bone marrow failure syndromes, and cancer susceptibility. Although various diagnostic tools are available to evaluate DNA damage, their quality to identify DNA repair deficiencies differs enormously and depends on affected pathways. In this study, we investigated the DDR biomarkers γH2AX (Ser139), p-ATM (Ser1981), and p-CHK2 (Thr68) using flow cytometry on peripheral blood cells obtained from patients with combined immunodeficiencies due to non-homologous end-joining (NHEJ) defects and ataxia telangiectasia (AT) in response to low-dose IR. Significantly reduced induction of all three markers was observed in AT patients compared to controls. However, delayed downregulation of γH2AX was found in patients with NHEJ defects. In contrast to previous reports of DDR in cellular models, these biomarkers were not sensitive enough to identify ARTEMIS deficiency with sufficient reliability. In summary, DDR biomarkers are suitable for diagnosing NHEJ defects and AT, which can be useful in neonates with abnormal TREC levels (T cell receptor excision circles) identified by newborn screening. We conclude that DDR biomarkers have benefits and some limitations depending on the underlying DNA repair deficiency.

scholarly journals A genetic map of the response to DNA damage in human cells

2019 ◽  
Author(s):  
Michele Olivieri ◽  
Tiffany Cho ◽  
Alejandro Álvarez-Quilón ◽  
Kejiao Li ◽  
Matthew J. Schellenberg ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase Ii ◽  
Dna Damage Response ◽  
Bone Marrow Failure ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Human Cells ◽  
Bone Marrow Failure Syndrome ◽  
Damage Response ◽  
Genotoxic Agents

SUMMARYThe response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

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