TiO2nanoparticles induce DNA double strand breaks and cell cycle arrest in human alveolar cells

2014 ◽  
Vol 56 (2) ◽  
pp. 204-217 ◽  
Author(s):  
Krupa Kansara ◽  
Pal Patel ◽  
Darshini Shah ◽  
Ritesh K. Shukla ◽  
Sanjay Singh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Alveolar Cells ◽  
Strand Breaks ◽  
Cycle Arrest

High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair

2003 ◽  
Vol 285 (2) ◽  
pp. F266-F274 ◽  
Author(s):  
Natalia I. Dmitrieva ◽  
Dmitry V. Bulavin ◽  
Maurice B. Burg
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Damage Response

High NaCl causes DNA double-strand breaks and cell cycle arrest, but the mechanism of its genotoxicity has been unclear. In this study, we describe a novel mechanism that contributes to this genotoxicity. The Mre11 exonuclease complex is a central component of DNA damage response. This complex assembles at sites of DNA damage, where it processes DNA ends for subsequent activation of repair and initiates cell cycle checkpoints. However, this does not occur with DNA damage caused by high NaCl. Rather, following high NaCl, Mre11 exits from the nucleus, DNA double-strand breaks accumulate in the S and G2 phases of the cell cycle, and DNA repair is inhibited. Furthermore, the exclusion of Mre11 from the nucleus by high NaCl persists following UV or ionizing radiation, also preventing DNA repair in response to those stresses, as evidenced by absence of H2AX phosphorylation at places of DNA damage and by impaired repair of damaged reporter plasmids. Activation of chk1 by phosphorylation on Ser345 generally is required for DNA damage-induced cell cycle arrest. However, chk1 does not become phosphorylated during high NaCl-induced cell cycle arrest. Also, high NaCl prevents ionizing and UV radiation-induced phosphorylation of chk1, but cell cycle arrest still occurs, indicating the existence of alternative mechanisms for the S and G2/M delays. DNA breaks that occur normally during processes such as DNA replication and transcription, as well as damages to DNA induced by genotoxic stresses, ordinarily are rapidly repaired. We propose that inhibition of this repair by high NaCl results in accumulation of DNA damage, accounting for the genotoxicity of high NaCl, and that cell cycle delay induced by high NaCl slows accumulation of DNA damage until the DNA damage-response network can be reactivated.

scholarly journals PS2 - 153 Dianhydrogalactitol (VAL-083) Treatment Causes Irreparable DNA Double-Strand Breaks, S/G2 Phase Cell-Cycle Arrest and Cell Death in Cancer Cells

2016 ◽  
Vol 43 (S4) ◽  
pp. S12-S13
Author(s):  
B. Zhai ◽  
A. Steino ◽  
J. Bacha ◽  
D. Brown ◽  
M. Daugaard
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Double Strand Breaks ◽  
Phase Cell ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest

Dianhydrogalactitol (VAL-083) is a unique bi-functional alkylating agent causing N7-guanine-methylation and inter-strand DNA crosslinks. VAL-083 readily crosses the blood-brain barrier, accumulates in brain tumor tissue and has shown activity in prior NCI-sponsored clinical trials against various cancers, including glioblastoma (GBM) and medulloblastoma. VAL-083 is also active against GBM cancer stem cells and acts as a radiosensitizer independent of O6-methylguanine-DNA methyltransferase activity (in contrast to e.g. temozolomide and BCNU). Here we report new insights into VAL-083 mechanism of action by showing that VAL-083 induces irreversible cell-cycle arrest and cell death caused by replication-dependent DNA damage. In lung (H2122, H1792, H23, A549) and prostate (PC3, LNCaP) cancer cell lines VAL-083 treatment caused irreversible S/G2 cell-cycle arrest and cell death (IC50 range 3.06-25.7 µM). VAL-083 pulse-treatment led to persistent phosphorylation of DNA double-strand breaks (DSB) sensors ATM, single-strand DNA-binding Replication Protein A (RPA32), and histone variant H2A.X, suggesting persistent DNA lesions. After 10 months in culture with increasing VAL-083 concentrations, H1792 and LNCaP cells survive at concentrations up to 9.4 µM and 7.4 µM, respectively, suggesting that efficient resistance mechanisms are not easily acquired by the cancer cells. Taken together with previous results showing that VAL-083 circumvents cisplatin-resistance and is less dependent on p53 activity than cisplatin, these results suggest a molecular mechanism for VAL-083 that differs from both TMZ, BCNU and cisplatin. They further suggest that irreparable DNA damage induced by VAL-083 is impervious to common strategies employed by cancer cells to escape effects of alkylating drugs used in GBM treatment.

Overexpression of EVI1 Causes Genomic Instability and Cell Cycle Arrest in Hematopoietic Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2398-2398
Author(s):  
Friederike Herbst ◽  
Claudia R Ball ◽  
Oksana Zavidij ◽  
Annika Mengering ◽  
Sylvia Fessler ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Genetic Instability ◽  
Transgene Expression ◽  
Hematopoietic Cells ◽  
Double Strand Breaks ◽  
Control Vector ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
G0 Phase

Abstract Abstract 2398 Deregulated expression of the zinc finger transcription factor ecotropic viral integration site 1 (EVI1) is an independent poor prognostic marker for patients with acute myeloid leukemia. Moreover, we have recently shown that in clinical gene therapy of chronic granulomatous disease (cGD) the activation of this gene locus by strong promoter and enhancer elements within the gamma-retroviral vector LTR has led to clonal dominance and malignant transformation. Physiologically, EVI1 is essential for embryonic development and in regulating hematopoietic stem cell self-renewal. As very little is known about its molecular mechanism driving hematopoietic transformation towards leukemia, we aim to systematically analyze the role of deregulated EVI1 expression and its larger splice form MDS1/EVI1 in hematopoiesis. Lentiviral vector particles encoding for EVI1 (E) or MDS1/EVI1 (ME) and eGFP as marker protein were produced to stably overexpress the transgenes. Transgene expression was verified in myeloid HL60 cells by western blotting and immunofluorescence staining. Analysis of growth kinetics of human HL60 cells in suspension cultures revealed 1.5 – 3 folds lower cell counts of ME and E expressing cells as compared to eGFP control vector transduced cells. We further analyzed the expression of transferrin receptor CD71 which is mainly expressed on proliferating cells to promote iron uptake. Both, E and ME transduced cells, down-regulated CD71 during seven days in culture as compared to eGFP-transduced control cells. 64.2 ± 0.4% of ME cells and 31.1 ± 1.6% of E cells expressed CD71 compared to untransduced (97.4 ± 0.0%) and control vector cells (94.8 ± 0.8%) (p<0.001). For further analyzing the transgene effect on cell cycle activity, 3 populations with different intensity of transgene expression (negative, intermediate and high eGFP+ cells) were isolated. With raising ME expression a 5-fold decrease in the percentage of cells in sub-G1 phase but a 1.3-fold increase in the percentage of cells in G1/G0 phase of the cell cycle was detected. In the highly EVI1 expressing fraction 91.4% arrested in G1/G0 phase of the cell cycle (50.4% in G1/G0 phase in eGFP− E cells). Moreover, almost 1/3 of these transcription factor expressing cells (29.8 ± 8.3% of ME and 27.2 ± 1.6% of EVI1 positive cells) could be detected in G0 phase as compared to 5.0 ± 1.3% of control vector transduced cells. In human CD34+ hematopoietic cells, E and ME overexpression led to a decrease of eGFP+ cells from 8% at day 3 after transduction to 0.5 – 2.5% at day 14 in suspension culture. In contrast, the proportion of eGFP+ human primary cells remained stable for the time period analyzed after transduction with the control vector. We then asked if the cell cycle arrest in G0/G1 is associated with genetic instability, as patients with insertional activation of EVI1 developed a myelodysplastic syndrome with monosomy 7. Analyzing global gene expression comparing mock and eGFP control vector transduced cells with EVI1 expressing hematopoietic cells revealed more than 2000 differentially regulated genes. Genes involved in cell cycle regulation, recombination, replication and DNA repair were significantly downregulated upon EVI1 expression compared to control cells. For further studying DNA damage repair capacity, we irradiated EVI1- and eGFP-control vector transduced cells. Staining of γ-H2AX, an indirect marker for DNA double strand breaks (DSB), revealed that EVI1+ γ-H2AX+ cells were enriched almost 2-fold in G0/G1 phase of the cell cycle as compared to control vector transduced cells. The number of DSB positive cells decreased within 6 hours without apoptosis indicating that most of the double-strand breaks were repaired by non-homologous end-joining. In summary, our data show that EVI1 overexpression causes G0 cell cycle arrest of hematopoietic cells possibly associated with genetic instability. DSB repair in EVI1+ cells may subsequently lead to the accumulation of additional mutations. Systematic investigation of EVI1 and MDS1/EVI1 overexpression in human hematopoietic cells will gain insights into mechanisms leading to clonal dominance and malignant transformation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutations in Saccharomyces cerevisiae That Block Meiotic Prophase Chromosome Metabolism and Confer Cell Cycle Arrest at Pachytene Identify Two New Meiosis-Specific Genes SAE1 and SAE3

Genetics ◽  
1997 ◽  
Vol 146 (3) ◽  
pp. 817-834 ◽  
Author(s):  
Andrew H Z McKee ◽  
Nancy Kleckner
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Meiotic Recombination ◽  
Meiotic Prophase ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Reading Frame ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Short Open Reading Frame

Two new meiosis-specific genes, SAE1 and SAE3, have been identified in a screen for mutations that confer an intermediate block in meiotic prophase. Such mutations confer a block to spore formation that is circumvented by addition of a mutation that eliminates meiotic recombination initiation and other aspects of chromosome metabolism, i.e., spo11. We show that sae1-1 and sae3-1 mutations each confer a distinct defect in meiotic recombination. sae1-1 produces recombinants but very slowly and ultimately to less than half the wild-type level; sae3-1 makes persistent hyper-resected meiotic double-strand breaks and has a severe defect in formation of recombinants. Both mutants arrest at the pachytene stage of meiotic prophase, sae1-1 temporarily and sae3-1 permanently. The phenotypes conferred by sae3-1 are similar to those conferred by mutation of the yeast RecA homologue DMC1, suggesting that SAE3 and DMC1 act at the same step(s) of chromosome metabolism. These results provide further evidence that intermediate blocks to prophase chromosome metabolism cause cell-cycle arrest. SAE1 encodes a 208-residue protein homologous to vertebrate mRNA cap-binding protein 20. SAE3 corresponds to a meiosis-specific RNA encoding an unusually short open reading frame of 50 codons.

Cells from ERCC1-deficient mice show increased genome instability and a reduced frequency of S-phase-dependent illegitimate chromosome exchange but a normal frequency of homologous recombination

1998 ◽  
Vol 111 (3) ◽  
pp. 395-404 ◽  
Author(s):  
D.W. Melton ◽  
A.M. Ketchen ◽  
F. Nunez ◽  
S. Bonatti-Abbondandolo ◽  
A. Abbondandolo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Genome Instability ◽  
Excision Repair ◽  
Ultra Violet ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Mutant Cells ◽  
Deficient Mice

The ERCC1 protein is essential for nucleotide excision repair in mammalian cells and is also believed to be involved in mitotic recombination. ERCC1-deficient mice, with their extreme runting and polyploid hepatocyte nuclei, have a phenotype that is more reminiscent of a cell cycle arrest/premature ageing disorder than the classic DNA repair deficiency disease, xeroderma pigmentosum. To understand the role of ERCC1 and the link between ERCC1-deficiency and cell cycle arrest, we have studied primary and immortalised embryonic fibroblast cultures from ERCC1-deficient mice and a Chinese hamster ovary ERCC1 mutant cell line. Mutant cells from both species showed the expected nucleotide excision repair deficiency, but the mouse mutant was only moderately sensitive to mitomycin C, indicating that ERCC1 is not essential for the recombination-mediated repair of interstrand cross links in the mouse. Mutant cells from both species had a high mutation frequency and the level of genomic instability was elevated in ERCC1-deficient mouse cells, both in vivo and in vitro. There was no evidence for an homologous recombination deficit in ERCC1 mutant cells from either species. However, the frequency of S-phase-dependent illegitimate chromatid exchange, induced by ultra violet light, was dramatically reduced in both mutants. In rodent cells the G1 arrest induced by ultra violet light is less extensive than in human cells, with the result that replication proceeds on an incompletely repaired template. Illegitimate recombination, resulting in a high frequency of chromatid exchange, is a response adopted by rodent cells to prevent the accumulation of DNA double strand breaks adjacent to unrepaired lesion sites on replicating DNA and allow replication to proceed. Our results indicate an additional role for ERCC1 in this process and we propose the following model to explain the growth arrest and early senescence seen in ERCC1-deficient mice. In the absence of ERCC1, spontaneously occurring DNA lesions accumulate and the failure of the illegitimate recombination process leads to the accumulation of double strand breaks following replication. This triggers the p53 response and the G2 cell cycle arrest, mediated by increased expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1). The increased levels of unrepaired lesions and double strand breaks lead to an increased mutation frequency and genome instability.

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.

scholarly journals Regulation of Chk2 Ubiquitination and Signaling through Autophosphorylation of Serine 379

2008 ◽  
Vol 28 (19) ◽  
pp. 5874-5885 ◽  
Author(s):  
Christine M. Lovly ◽  
Ling Yan ◽  
Christine E. Ryan ◽  
Saeko Takada ◽  
Helen Piwnica-Worms
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Ionizing Radiation ◽  
Kinase Activity ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Autophosphorylation Site

ABSTRACT The Chk2 protein kinase protects genome integrity by promoting cell cycle arrest or apoptosis in response to DNA double-strand breaks, and Chk2 mutations are found in both familial and sporadic cancers. Exposure of cells to ionizing radiation (IR) or radiomimetic drugs induces Chk2 phosphorylation by ATM, followed by Chk2 oligomerization, auto-/transphosphorylation, and activation. Here we demonstrate that Chk2 is ubiquitinated upon activation and that this requires Chk2 kinase activity. Serine 379 (S379) was identified as a novel IR-inducible autophosphorylation site required for ubiquitination of Chk2 by a Cullin 1-containing E3 ligase complex. Importantly, S379 was required for Chk2 to induce apoptosis in cells with DNA double-strand breaks. Thus, auto-/transphosphorylation of S379 is required for Chk2 ubiquitination and effector function.

scholarly journals The Functions of Budding Yeast Sae2 in the DNA Damage Response Require Mec1- and Tel1-Dependent Phosphorylation

2004 ◽  
Vol 24 (10) ◽  
pp. 4151-4165 ◽  
Author(s):  
Enrico Baroni ◽  
Valeria Viscardi ◽  
Hugo Cartagena-Lirola ◽  
Giovanna Lucchini ◽  
Maria Pia Longhese
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Synthetic Lethality ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Biological Responses

ABSTRACT DNA damage checkpoint pathways sense DNA lesions and transduce the signals into appropriate biological responses, including cell cycle arrest, induction of transcriptional programs, and modification or activation of repair factors. Here we show that the Saccharomyces cerevisiae Sae2 protein, known to be involved in processing meiotic and mitotic double-strand breaks, is required for proper recovery from checkpoint-mediated cell cycle arrest after DNA damage and is phosphorylated periodically during the unperturbed cell cycle and in response to DNA damage. Both cell cycle- and DNA damage-dependent Sae2 phosphorylation requires the main checkpoint kinase, Mec1, and the upstream components of its pathway, Ddc1, Rad17, Rad24, and Mec3. Another pathway, involving Tel1 and the MRX complex, is also required for full DNA damage-induced Sae2 phosphorylation, that is instead independent of the downstream checkpoint transducers Rad53 and Chk1, as well as of their mediators Rad9 and Mrc1. Mutations altering all the favored ATM/ATR phosphorylation sites of Sae2 not only abolish its in vivo phosphorylation after DNA damage but also cause hypersensitivity to methyl methanesulfonate treatment, synthetic lethality with RAD27 deletion, and decreased rates of mitotic recombination between inverted Alu repeats, suggesting that checkpoint-mediated phosphorylation of Sae2 is important to support its repair and recombination functions.

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