ANKHD1 Silencing Delays S Phase Progression in Multiple Myeloma Cells Via Activation of ATM/ATR -CDC25a Pathway

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5624-5624
Author(s):  
Dhyani Anamika ◽  
Patricia Favaro ◽  
Sara Teresinha Olalla Saad
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
S Phase ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Myeloma Cells ◽  
Checkpoint Kinase ◽  
Strand Breaks ◽  
Phase Progression

Abstract Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells. In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point. References: 1) ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells. Dhyani et al. FEBS letters 2012; 586: 4311-18. 2) ANKHD1 is essential for repair of DNA double strand breaks in multiple myeloma. Dhyani et al. ASH Abstract, Blood 2015; 126:1762. Disclosures No relevant conflicts of interest to declare.

ANKHD1 Is Essential for Repair of DNA Double-Strand Breaks in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1762-1762
Author(s):  
Anamika Dhyani ◽  
Patricia Favaro ◽  
Sara T. Olalla Saad
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Replication ◽  
S Phase ◽  
Histone Gene ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Replication And Repair ◽  
Histone Gene Transcription

Abstract ANKHD1, Ankyrin repeat and KH domain-containing protein is highly expressed and plays an important role in the proliferation and cell cycle progression of multiple myeloma (MM) cells. Inhibition of ANKHD1 expression upregulates p21 (CDKN1A, Cyclin Dependent Kinase Inhibitor), a potent cell cycle regulator, and its expression represses p21 promoter. Upregulation of p21 was found to be irrespective of the TP53 mutational status of MM cell lines. A study by our group has shown that ANKHD1 is highly expressed in S phase and that the inhibition of ANKHD1 expression downregulates replication dependent histones suggesting that it might be required for histone transcription (1). Assuming that ANKHD1 might be involved in the transcripitional activation of histones, we studied the effect of ANKHD1 silencing on nuclear protein of the ataxia telangiectasia mutated locus (NPAT), a component of the cell-cycle-dependent histone gene transcription machinery. NPAT associates with histone gene promoters in S phase and suppression of NPAT expression impedes expression of all histone subtypes. In present study, there was a decreased expression of NPAT in ANKHD1 silenced MM cells. Despite the fact that both ANKHD1 and NPAT were localized in the nucleus of MM cells, they did not appear to associate, as observed by confocal microscopy, suggesting at present that ANKHD1 does not modulate histones via NPAT. Since DNA replication is coupled with histone synthesis and downregulation of histones is associated with replication stress and DNA damage, we checked for expression of PCNA (Proliferating Cell Nuclear Antigen), protein involved in DNA replication and repair. PCNA expression was found to be significantly decreased in ANKHD1 inhibited MM cells, suggesting its role in PCNA mediated DNA replication and repair (Fig. 1). To confirm this, we studied the effect of ANKHD1 silencing on some of the components of DNA damage repair (DDR) pathway. We observed increased expression of gamma- H2AX (γ-H2AX i.e Phosphorylated Histone H2AX), marker for DNA double-strand breaks (DSBs) and an early sign of DNA damage induced by replication stress (Fig. 1). We also observed a decrease in phosphorylated CHK2 (Check Point Kinase 2), an essential serine threonine kinase involved in DDR. Accumulation of γ-H2AX on ANKHD1 silencing confirms DNA damage and suggests the possible mechanism of ANKHD1 mediated histones downregulation. In summary, ANKHD1 silencing in MM cells leads to DNA damage (DSBs), suggesting that ANKHD1 is essential for DNA replication and repair. Furthermore, as ANKHD1 negatively regulates p21, and p21 controls DNA replication and repair by interacting with PCNA, we hypothesize that ANKHD1 might be playing role in DNA repair via modulation of the p21-PCNA pathway. Results of the role of ANKHD1 in DNA repair are however preliminary and need to be explored. References: 1) ANKHD1 Is Required for S Phase Progression and Histone Gene Transcription in Multiple Myeloma. Dhyani et al. ASH Abstract; Blood 2014. Figure 1. Western blot analysis of proteins: a) PCNA and b) γ-H2AX, in control and ANKHD1 silenced U266 MM cell line. Tubulin and GAPDH were used as endogenous controls. Figure 1. Western blot analysis of proteins: a) PCNA and b) γ-H2AX, in control and ANKHD1 silenced U266 MM cell line. Tubulin and GAPDH were used as endogenous controls. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of ATR Overcomes Chemotherapy Resistance in p53 Deficient Myeloma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3109-3109
Author(s):  
Louise Bouard ◽  
Benoit Tessoulin ◽  
Géraldine Descamps ◽  
Cyrille Touzeau ◽  
Philippe Moreau ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Dna Repair ◽  
Chemotherapy Resistance ◽  
S Phase ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Myeloma Cells ◽  
Strand Breaks ◽  
Cycle Arrest

In MM, as well as in most hematological malignancies, deficiency in p53 pathway (mainly because of TP53 deletion and/or mutation) is associated with resistance to treatments (Tessoulin Blood Reviews 2017; 31:251). Recent clinical studies have shown that deletion or mutation of TP53 are the most adverse prognostic values for patients (Thakurta Blood 2019;133:1217). Although these patients are easily identified, there is no dedicated therapies for them. p53 pathway is central for homeostasis and cell adaptation/response to many stresses, including DNA repair orchestration and survival regulation. In p53 deficient cells, DNA damaging drugs don't induce massive apoptosis and cells escape to death. In normal cells, DNA damaging drugs induce cell cycle arrest and DNA repair, mainly orchestrated by p53 target genes. Cell cycle arrest in S phase, which is critical for allowing homologous DNA repair, is activated by cell cycle check-point inhibitor such as Chk1, an ATR target. In p53 deficient cells, inhibiting check point inhibitor using ATR inhibitor should allow DNA damaged cells to progress into cell cycle despite the lack of repair and in fine induce replicative/mitotic catastrophe. The aim of this study was to assess whether inhibiting ATR in p53 deficient myeloma cells could overcome chemotherapy resistance. ATR inhibitor, VE-821, was assessed in 13 human myeloma cell lines (HMCLs) alone and in combination with DNA damaging agents, CX5461, a G quadruplex inhibitor, or melphalan, the « myeloma » alkylating drug. The HMCL cohort included 8 HMCLs, 5 TP53Abn and 5 TP53wt. Cell viability was assessed using Cell Titer Glo assay or using flow cytometry (loss of AnnexinV or CD138 staining in HMCLs or primary myeloma cells, respectively). In our cohort of 13 HMCLs and by contrast to previous results, CX5461 was more efficient in TP53wt than in TP53abn HMCLs (mean of death at 0.5mM was 43% versus 24%, p=0.04). Melphalan was also more potent in TP53wt than in TP53abn HMCLs (LD50 values were 26 mM versus 10 mM, p=0.008). By contrast, ATR inhibitor VE-821 (2.5mM) was efficient in both types of HMCLs (mean of death in TP53wt was 45% and 28% in TP53abn HMCLs, p=0.6). Combination of CX5641 (0.5mM) with VE-821 (2.5mM) was more efficient than each drug alone and efficacy was not dependent on TP53 status (mean of death in TP53wt was 69% versus 56% in TP53abn HMCLs, p=0.6): interestingly, combo was efficient in all TP53abn HMCLs, being either additive (n=5) or even synergistic (n=3). By contrast, combo was not efficient in all TP53wt HMCLs (either additive or antagonist). Combination of melphalan (10 mM) with VE-821 (2.5mM) was also synergistic in TP53abn HMCLs (mean of cell death was 9% with melphalan and 73% for combo, p<0.05). Preliminary results of combos in 6 consecutive primary samples with MM or plasma cell leukemia (3 TP53wt and 3 TP53abn) demonstrated efficacy. Indeed, in the 3 TP53abn samples, both CX5641/VE-821 and melphalan/VE-821 combos displayed synergism or additivity: median of expected values versus observed values was 61% versus 74% for CX5641/VE-821, and 98% versus 89% for melphalan/VE-821, respectively. In the 3 TP53wt samples, combos displayed additivity or antagonism: median of expected versus observed values was 15% versus 15% for CX5641/VE-821, and 100% versus 62% for melphalan/VE-821, respectively. In normal peripheral blood cells (n=2), both combos were not cytotoxic (mean values of cell death were 0% with CX5641/VE-821 and 3% with melphalan/VE-821). To decipher the molecular pathway involved in cell response, we monitored cell cycle using BrdU/IP assay, replicative stress response using Chk1 phosphorylation and DNA double strand breaks using Comets assays in 3 TP53abn HMCLs. At 24h, CX5641 induced an increase of cells in S (mean of increase 12%) and G2M phases (11%), while VE-821 didn't modify cell cycle. Combination of CX5641 with VE-821 induced a dramatic increase of cells in G2M (20%) (and in subG2 phase), and a decrease of cells in S phase (10%), indicating that cells blocked in S phase by CX5641 were released by VE-821.CX5641 induced Chk1 phosphorylation, which was inhibited by addition of VE-821, confirming the CX5641/ATR/Chk1 signaling. Finally, CX5641 and VE-821 induced comets, confirming irreversible DNA double strand breaks. All these results show that inhibition of ATR after inducing DNA damage in TP53abn myeloma cells efficiently induces cell death, while preserving normal cells. Disclosures Moreau: Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Loss Of NIPA Leads To Defects In The Repair Of DNA Double Strand Breaks In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2488-2488
Author(s):  
Anna Lena Illert ◽  
Cristina Antinozzi ◽  
Hiroyuki Kawaguchi ◽  
Michal Kulinski ◽  
Christine Klitzing ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Meiotic Prophase ◽  
Conflicts Of Interest ◽  
Cyclin B1 ◽  
Double Strand Breaks ◽  
Conditional Knockout ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Chromosome Axis

Abstract Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding Nipa. Nipa-deficient animals are viable, but show a lower birth rate and a reduced body weight. Furthermore, Nipa-deficient males were sterile due to a block of spermatogenesis during meiotic prophase. Virtually no spermatocytes progress beyond a late-zygotene to early-pachytene stage and reach an aberrant stage, with synaptonemal complex disassembly and incomplete synapsis. Nipa-/- females are sub-fertile with an early and severe meiotic defect during embryogenesis with extensive apoptosis in early prophase (E13.5-E14.5). Here we report, that Nipa-/- meiocytes exhibit persistent cytological markers for DNA double strand break repair proteins (like DMC1, RAD51) in meiotic prophase with more than twice as many DMC1 foci as control animals. Kinetic analysis of the first wave of spermatogenesis showed increased DMC1/RAD51 foci in Nipa-/- cells as soon as early-pachynema cells appear (13-14 days post partum). Moreover, we show that Nipa deficiency does not lead to a defect in meiotic sex chromosome inactivation despite epithelial stage IV apoptosis. Nipa-deficient spermatocytes exhibit numerous abnormalities in staining of chromosome axis associated proteins (like SYCP3 and STAG3) indicating that chromosome axis defects were associated with compromised chromosome axis integrity leading to overt chromosome fragmentation. Further in vitro analyses with bleomycin treated MEFs displayed high pH2AX levels in cells lacking NIPA. Repair of DNA DSB seemed to be abolished in these cells as the pH2AX-level were sustained and still visible after 90 min of timecourse, where wildtype cells already repaired sides of DNA Damage. Consistent with these findings NIPA-deficient spleen cells showed compromised DNA Damage repair measured in a comet assay with a significantly longer olive tail moment in NIPA knockout cells under repair conditions. Taken together, the phenotype of Nipa-knockout mice is a definitive proof of the meiotic significance of NIPA and our results show a new, unsuspected role of NIPA in chromosome stability and the repair of DNA double strand breaks. Disclosures: No relevant conflicts of interest to declare.

scholarly journals SET8 Is a Potential Therapeutic Target in MM

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4435-4435
Author(s):  
Herviou Laurie ◽  
Fanny Izard ◽  
Elke De Bruyne ◽  
Eva Desmedt ◽  
Anqi Ma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Myeloma Cells ◽  
Strand Breaks ◽  
Damage Response

Abstract Epigenetic regulation mechanisms - such as histone marks, DNA methylation and miRNA - are often misregulated in cancers and are associated with tumorigenesis and drug resistance. Multiple Myeloma (MM) is a malignant plasma cell disease that accumulates within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with resistance to chemotherapy. This epigenetic plasticity can be targeted with epidrugs, nowadays used in treatment of several cancers. We recently identified a significant overexpression of the lysine histone methyltransferase SETD8 in MM cells (HMCLs; N=40) compared with normal plasma cells (N=5) (P<0.001). SETD8 (also known as SET8, PR-Set7, KMT5A) is the sole enzyme responsible for the monomethylation of histone H4 at lysine 20 (H4K20me1) which has been linked to chromatin compaction and cell-cycle regulation. In addition, SETD8 induces the methylation of non-histone proteins, such as the replication factor PCNA, the tumor suppressor P53 and its stabilizing protein Numb. While SETD8-mediated methylation of P53 and Numb inhibits apoptosis, PCNA methylation upon SETD8 enhances the interaction with the Flap endonuclease FEN1 and promotes cancer cell proliferation. SETD8 is also implicated in DNA damage response, helping 53BP1 recruitment at DNA double-strand breaks. Consistent with this, overexpression of SETD8 is found in various types of cancer and has been directly implicated in breast cancer invasiveness and metastasis. A role of SETD8 in development of MM has however never been described. We found that high SETD8 expression is associated with a poor prognosis in 2 independent cohorts of newly diagnosed patients (UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N=158). Specific SETD8 inhibition with UNC-0379 inhibitor, causing its degradation and H4K20me1 depletion, leads to significant growth inhibition of HMCLs (N=10) and the murine cell lines 5T33MM and 5TGM1. MM cells treated with UNC-0379 presented a G0/G1 cell cycle arrest after 24h of treatment, followed by apoptosis 48h later. To confirm that SETD8 inhibition is as efficient on primary MM cells from patients, primary MM cells (N=8) were co-cultured with their bone marrow microenvironment and recombinant IL-6 and treated for 4 days with UNC-0379. Interestingly, treatment of MM patient samples with UNC-0379 reduces the percentage of myeloma cells (65%; P<0.005) without significantly affecting the non-myeloma cells, suggesting a specific addiction of primary myeloma cells to SETD8 activity. Melphalan is an alkylating agent commonly used in MM treatment. As SETD8 is known to be involved in the DNA damage response, we investigated the effect of its combination with Melphalan on HMCLs. Results show that this particular drug combination strongly enhances double strand breaks in HMCLs monitored using 53BP1 foci formation and gH2AX detection. This result emphasizes a potential role of SETD8 in DNA repair in MM cells. Furthermore, GSEA analysis of patients with high SETD8 expression highlighted a significant enrichment of genes involved in DNA repair, MYC-MAX targets and MAPK pathway. Our study is the first to demonstrate the importance of SETD8 for MM cells survival and suggest that SETD8 inhibition represent a promising strategy to improve conventional treatment of MM with DNA damaging agents. Disclosures No relevant conflicts of interest to declare.

BCR/ABL Oncogenic Kinase Promotes Unfaithful Repair of the Reactive Oxygen Species - Dependent DNA Double-Strand Breaks.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 712-712 ◽  
Author(s):  
Tomasz Skorski ◽  
Michal O. Nowicki ◽  
Rafal Falinski ◽  
Mateusz Koptyra ◽  
Artur Slupianek ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Cycle ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Double Strand Breaks ◽  
Transformed Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Abl Kinase

Abstract The oncogenic BCR/ABL tyrosine kinase induces constitutive DNA damage in Philadelphia chromosome (Ph1)-positive leukemia cells. We find that BCR/ABL kinase - induced reactive oxygen species (ROS) cause chronic oxidative DNA damage as indicated by an enzymatic assay detecting oxidized bases. These DNA lesions result in DNA double-strand breaks (DSBs) detected by comet assay, immunofluorescent gamma-H2AX nuclear foci and linker-ligation PCR (LL-PCR). Combined analysis of the length of LL-PCR products and the sequences of two reference genes DR-GFP and Na+/K+ ATPase revealed that ROS dependent DSBs occurred in the regions containing multiple, 5–9bp long stretches of G/C, in concordance with the notion that oxidative DNA damage is predominantly detected in G/C-rich sequences. Elevated numbers of DSBs were detected in BCR/ABL cell lines, murine bone marrow cells transformed with BCR/ABL and in CML patient samples, in comparison to normal counterparts. Inhibition of the BCR/ABL kinase by STI571 and diminishion of ROS activity by the ROS scavenger PDTC reduced DSBs formation. Cell cycle analysis revealed that most of these DSBs occur during S and G2/M phases, and are probably associated with the stalled replication forks. Homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) represent two major mechanisms of DSBs repair in S and G2/M cell cycle phase. Using the in vivo recombination assay consisting of the DSB-dependent reconstitution of the green fluorescent protein (GFP) gene we found that HRR is stimulated in BCR/ABL-positive cells. In addition, in vitro assay measuring the activity of NHEJ revealed that this repair process is also activated by the BCR/ABL kinase. RAD51 and Ku70 play a key role in HRR and NHEJ, respectively. The reaction sites of HRR and NHEJ in the nuclei could be visualized by double-immunofluorescence detecting co-localization of gamma-H2AX foci (DSBs sites) with RAD51 (HRR sites) or Ku70 (NHEJ sites). Equal co-localization frequency of gamma-H2AX foci with RAD51 and Ku70 was detected, suggesting that both HRR and NHEJ play an important role in reparation of ROS-dependent DSBs in BCR/ABL-transformed cells. Analysis of the DSBs repair products in the reporter DR-GFP gene in BCR/ABL cells identified ~40% of HRR and ~60% of NHEJ events. Sequencing revealed point-mutations in HRR products and large deletions in NHEJ products in BCR/ABL-positive cells, but not in non-transformed cells. We propose that the following series of events may contribute to genomic instability of Ph1-positive leukemias: BCR/ABL → ROS → oxidative DNA damage → DSBs in proliferating cells → unfaithful HRR and NHEJ repair. Since BCR/ABL share many similarities with other members of the fusion tyrosine kinases (FTKs) family, these events may contribute to genomic instability of hematological malignancies caused by FTKs.

Hematopoietic Stem Cells Repair Ionizing Radiation-Induced DNA Double Strand Breaks in a Cell Cycle-Dependent Manner.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3241-3241
Author(s):  
Senthil Kumar Pazhanisamy ◽  
Ningfei An ◽  
Yong Wang ◽  
Daohong Zhou
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Adult Mouse ◽  
Double Strand Breaks ◽  
Hematopoietic Progenitor Cells ◽  
Dna Double Strand Breaks ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Strand Breaks ◽  
Quiescent Hscs

Abstract Abstract 3241 Poster Board III-178 Mice with mutations in various DNA repair genes exhibit accelerated aging due to hematopoietic stem cell (HSC) premature exhaustion, indicating that DNA repair is crucial for the maintenance of HSC self-renewal and hematopoietic function. In addition, some of these mutated mice are highly susceptible to the development of leukemia and lymphoma due to an increase in genomic instability in HSCs. However, how HSCs respond to genotoxic stress and repair DNA damage have not been well established and thus, were investigated in the present study using a mouse model. Specifically, DNA damage and repair were analyzed by gH2AX immunofluorescent staining and neutral comet assay to quantify IR-induced DNA double strand breaks (DSBs) in HSCs (Lin- c-kit+ Sca1+ cells or LKS+ cells) and hematopoietic progenitor cells (HPCs; Lin- c-kit+ Sca1- cells or LKS- cells) isolated from adult mouse bone marrow (BM) after they were exposed to ionizing radiation (IR). The results showed that exposure to IR induced a similar number of DSBs in HSCs and hematopoietic progenitor cells (HPCs) isolated from adult mouse BM. However, HPCs repaired the damage within 6 h after IR, whereas more than 50% DSBs were unrepaired by HSCs even at 24h after IR, indicating that HSCs are highly deficient in repair of IR-induced DSBs. The deficient DSBs repair in HSCs is attributable to their quiescence, as sorted quiescent Pyronin Ylow HSCs were more deficient in repairing the damage than cycling Pyronin Yhigh HSCs. This suggestion is further supported by the observations that proliferating HSCs such as fetal liver HSCs and HSCs isolated from 5-FU-treated adult mouse BM repaired the damage as efficiently as HPCs. In addition, incubation of quiescent Pyronin Ylow HSCs from adult BM with stem cell factor and thrombopoietin for 48 h stimulated the cell cycle entry and DNA damage repair function. These findings indicate that stimulation of cell cycling can promote HSCs to repair DNA damage. The difference in repair of IR-induced DSBs between quiescent and cycling HSCs is not because they express different levels of key proteins (such as Ku70, Ku80, DNA-PKcs, Lig4, XRCC4, Dclre1c, Nhej1, Brac-1, Brac-2, MRE11a, Nbs1, Rad50, Rad51, and ATM) involved in non-homologous end joining (NHEJ) and homologous recombination (HR). Instead, quiescent HSCs exhibited an insignificant activation of DNA-PK and minimal formation of XRCC4 and Rad51 foci after exposure to IR, suggesting that quiescent HSCs are deficient in DSBs repair through the NHEJ and HR pathways. However, quiescent HSCs exhibited similar levels of phosphorylation of ATM and p53 after IR compared to cycling HSCs and HPCs, indicating that quiescent HSCs are proficient in sensing DNA damage to initiate DNA damage responses. These findings provide crucial insights into how HSCs respond to and repair DNA damage, which could significantly advance our understanding on how HSCs maintain their genomic stability. Disclosures No relevant conflicts of interest to declare.

scholarly journals DNA damage triggers increased mobility of chromosomes in G1-phase cells

2019 ◽  
Vol 30 (21) ◽  
pp. 2620-2625 ◽  
Author(s):  
Michael J. Smith ◽  
Eric E. Bryant ◽  
Fraulin J. Joseph ◽  
Rodney Rothstein
Keyword(s):  
Dna Damage ◽  
S Phase ◽  
Double Strand Breaks ◽  
Homology Search ◽  
G1 Phase ◽  
Dna Damage Checkpoint ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Global Mobility ◽  
Break Site

During S phase in Saccharomyces cerevisiae, chromosomal loci become mobile in response to DNA double-strand breaks both at the break site (local mobility) and throughout the nucleus (global mobility). Increased nuclear exploration is regulated by the recombination machinery and the DNA damage checkpoint and is likely an important aspect of homology search. While mobility in response to DNA damage has been studied extensively in S phase, the response in interphase has not, and the question of whether homologous recombination proceeds to completion in G1 phase remains controversial. Here, we find that global mobility is triggered in G1 phase. As in S phase, global mobility in G1 phase is controlled by the DNA damage checkpoint and the Rad51 recombinase. Interestingly, despite the restriction of Rad52 mediator foci to S phase, Rad51 foci form at high levels in G1 phase. Together, these observations indicate that the recombination and checkpoint machineries promote global mobility in G1 phase, supporting the notion that recombination can occur in interphase diploids.

scholarly journals JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 709-718 ◽  
Author(s):  
Tanyel Kiziltepe ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Enrique M. Ocio ◽  
Noopur Raje ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Endonuclease G ◽  
Human Multiple Myeloma

Abstract Here we investigated the cytotoxicity of JS-K, a prodrug designed to release nitric oxide (NO•) following reaction with glutathione S-transferases, in multiple myeloma (MM). JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient-derived MM cells. JS-K induced apoptosis in MM cells, which was associated with PARP, caspase-8, and caspase-9 cleavage; increased Fas/CD95 expression; Mcl-1 cleavage; and Bcl-2 phosphorylation, as well as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) release. Moreover, JS-K overcame the survival advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells. Mechanistic studies revealed that JS-K–induced cytotoxicity was mediated via NO• in MM cells. Furthermore, JS-K induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by neutral comet assay, as well as H2AX, Chk2 and p53 phosphorylation. JS-K also activated c-Jun NH2-terminal kinase (JNK) in MM cells; conversely, inhibition of JNK markedly decreased JS-K–induced cytotoxicity. Importantly, bortezomib significantly enhanced JS-K–induced cytotoxicity. Finally, JS-K is well tolerated, inhibits tumor growth, and prolongs survival in a human MM xenograft mouse model. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.

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