Endosulfan causes the alterations of DNA damage response through ATM-p53 signaling pathway in human leukemia cells

2018 ◽  
Vol 238 ◽  
pp. 1048-1055 ◽  
Author(s):  
Dan Xu ◽  
Dong Liang ◽  
Yubing Guo ◽  
Yeqing Sun
Keyword(s):  
Dna Damage ◽  
Signaling Pathway ◽  
Dna Damage Response ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
P53 Signaling ◽  
Human Leukemia Cells ◽  
Damage Response ◽  
P53 Signaling Pathway

scholarly journals The SF3B1 K700E Mutation Induces R-Loop Accumulation and Associated DNA Damage

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4219-4219 ◽  
Author(s):  
Shalini Singh ◽  
Doaa Ahmed ◽  
Hamid Dolatshad ◽  
Dharamveer Tatwavedi ◽  
Ulrike Schulze ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
K562 Cells ◽  
Splicing Factor ◽  
Leukemia Cells ◽  
Cd34 Cells ◽  
Damage Response ◽  
Healthy Control ◽  
Atr Activation ◽  
Mutant Cells

The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1 and ZRSR2) are the most common mutations found in MDS. There is evidence that some spliceosomal components play a role in the maintenance of genomic stability. Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of R-loops (RNA-DNA hybrids with a displaced single stranded DNA). Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loops formation in leukemia cell lines, resulting in increased DNA damage, replication stress and activation of the ATR-Chk1 pathway. SF3B1 is the most frequently mutated splicing factor gene in MDS, but a role for mutated SF3B1 in R-loop accumulation and DNA damage has not yet been reported in hematopoietic cells. We have investigated the effects of the common SF3B1 K700E mutation on R-loop formation and DNA damage response in MDS and leukemia cells. R-loop signals and the DNA damage response were measured by immunofluorescence staining using S9.6 and anti-γ-H2AX antibodies respectively. Firstly, we studied K562 (myeloid leukemia) cells with the SF3B1 K700E mutation and isogenic SF3B1 K700K wildtype (WT) K562 cells. K562 cells with SF3B1 mutation showed a significant increase in the number of S9.6 foci [Fold change (FC) 2.01, p<0.001] and in the number of γ-H2AX foci (FC 2.32, p<0.001), indicating increased R-loops and DNA damage, compared to SF3B1 WT K562 cells. Moreover, we observed increased Chk1 phosphorylation at Ser345, a hallmark of activation of the ATR pathway, in SF3B1 mutant K562 cells. Next, we analyzed induced pluripotent stem cells (iPSCs) that we generated from the bone marrow cells of one SF3B1 mutant MDS patient and of one healthy control. A significant increase in R-loops and DNA damage response was observed in an iPSC clone harboring SF3B1 mutation compared to another iPSC clone without SF3B1 mutation obtained from same MDS patient (S9.6 mean fluorescence intensity - FC 1.72, p<0.001; γ-H2AX foci - FC 1.34, p=0.052) and to iPSCs from the healthy control (S9.6 mean fluorescence intensity - FC 1.53, p<0.001; γ-H2AX foci - FC 1.61, p=0.006). In addition, bone marrow CD34+ cells from a SF3B1 mutant MDS patient showed increased R-loops (as measured by number of S9.6 foci) compared to CD34+ cells from a MDS patient without splicing factor mutations (FC 1.9) and from a healthy control (FC 2.6). To investigate whether the observed DNA damage and ATR activation in SF3B1 mutant K562 cells result from induced R-loops, we overexpressed RNASEH1 (encoding an enzyme that degrades the RNA in RNA:DNA hybrids) to resolve R-loops in these cells. RNASEH1 overexpression significantly reduced the number of S9.6 (FC 0.51, p<0.001) and γ-H2AX foci (FC 0.63, p=0.035) in SF3B1 mutant K562 cells compared to SF3B1 WT K562 cells. RNASEH1 overexpression also resulted in decreased Chk1 phosphorylation, indicating suppression of ATR pathway activation in SF3B1 mutant K562 cells. To determine the functional importance of ATR activation associated with SF3B1 mutation, we evaluated the sensitivity of SF3B1 mutant cells towards the ATR inhibitor VE-821. SF3B1 mutant K562 cells showed preferential sensitivity towards VE-821 compared to SF3B1 WT K562 cells. Chk1 is a critical substrate of ATR, and we next investigated the effects of Chk1 inhibition in SF3B1 mutant cells. Interestingly, SF3B1 mutant K562 cells demonstrated preferential sensitivity towards the Chk1 inhibitor UCN-1 (IC50 61.8 nM) compared to SF3B1 WT K562 cells (IC50 267 nM), suggesting that ATR activation is important for the survival of SF3B1 mutant cells. SF3B1 mutant K562 cells were preferentially sensitive to the splicing modulator Sudemycin D6 (IC50 53.2 nM) compared to SF3B1 WT K562 cells (IC50 130.7 nM). The effects of VE-821 and UCN-1 on SF3B1 mutant K562 cells were enhanced by Sudemycin D6 (Combination index <1), indicating synergy. In summary, our results show that the SF3B1 mutation leads to accumulation of R-loops and associated DNA damage resulting in activation of the ATR pathway in MDS and leukemia cells. Thus different mutated splicing factors have convergent effects on R-loop elevation leading to DNA damage. Moreover, our data suggest that Chk1 inhibition, alone or in combination with splicing modulators, may represent a novel therapeutic strategy to target SF3B1 mutant cells. Disclosures Schuh: Janssen: Speakers Bureau; Verastem: Speakers Bureau; Kite: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Wiseman:Novartis, Celgene: Consultancy, Honoraria.

scholarly journals INTS3 controls the hSSB1-mediated DNA damage response

2009 ◽  
Vol 187 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Jeffrey R. Skaar ◽  
Derek J. Richard ◽  
Anita Saraf ◽  
Alfredo Toschi ◽  
Emma Bolderson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Signaling Pathway ◽  
Dna Damage Response ◽  
Ataxia Telangiectasia ◽  
Binding Protein ◽  
Ataxia Telangiectasia Mutated ◽  
Uncharacterized Protein ◽  
Damage Response ◽  
Atm Signaling Pathway ◽  
Atm Signaling

Human SSB1 (single-stranded binding protein 1 [hSSB1]) was recently identified as a part of the ataxia telangiectasia mutated (ATM) signaling pathway. To investigate hSSB1 function, we performed tandem affinity purifications of hSSB1 mutants mimicking the unphosphorylated and ATM-phosphorylated states. Both hSSB1 mutants copurified a subset of Integrator complex subunits and the uncharacterized protein LOC58493/c9orf80 (henceforth minute INTS3/hSSB-associated element [MISE]). The INTS3–MISE–hSSB1 complex plays a key role in ATM activation and RAD51 recruitment to DNA damage foci during the response to genotoxic stresses. These effects on the DNA damage response are caused by the control of hSSB1 transcription via INTS3, demonstrating a new network controlling hSSB1 function.

Dysregulation, But Not Mutation Of p53 Signaling Pathway In Breast Implant-Associated Anaplastic Large Cell Lymphoma Cell Lines

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4879-4879 ◽  
Author(s):  
Hai Wang ◽  
Chao Xie ◽  
Shiwu Li ◽  
Eva V. George ◽  
Westley H. Reeves ◽  
...  
Keyword(s):  
Dna Damage ◽  
Signaling Pathway ◽  
Cell Lines ◽  
P53 Protein ◽  
Breast Implant ◽  
Large Cell ◽  
Qrt Pcr ◽  
P53 Signaling ◽  
P53 Activation ◽  
P53 Signaling Pathway

Abstract A consistent feature of over 100 reported cases of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is their complex cytogenetic abnormalities, suggesting that genomic instability may drive lymphomagenesis and/or tumor progression. Loss of heterozygosity(LOH) of the TP53 tumor suppressor gene locus on the short arm of chromosome 17 (17p13.1) is a frequent finding. Human p53 plays an important role in cell cycle arrest, DNA repair, and apoptosis and it maintains genome stability by preventing mutations. Recently, three T cell breast lymphoma (TLBR) cell lines were derived from patients’ BIA-ALCL primary tumor biopsy specimens. These cell lines are IL-2 dependent, ALK-negative, CD30+activated cytotoxic T cells closely resembling the original tumor cells. Thus, the cell lines may serve as an important tool for studying this newly recognized disease entity. Because of its rarity, the clinical pathologic features, tumor cell biology, and genetics of BIA-ALCL have yet to be fully defined. Here we tested the hypothesis that the p53 signaling pathway is defective in TLBR cells. We initially examined TP53 transcript expression among the cell lines. By qRT-PCR, p53 transcripts were detected in all three lines, with the highest level in TLBR-2. Next we examined p53 protein expression and p53 activation in response to ultraviolet (UV) or gamma irradiation. By Western blotting, all TLBR cell lines expressed much lower levels of p53 protein following UV irradiation (400 J/m2) than Karpas (ALK+ ALCL) cells and failed to show ATM/ATR-induced phosphorylation of p53 on serine 15, an early indicator of p53 activation. Genetic defects (deletion, mutation) of the p53 coding sequence were not found by Sanger sequencing. Interestingly, a polymorphism at p53 codon 72 (Arg72Pro), a normal variant associated with increased susceptibility to breast cancer, was detected in TLBR-1 and -3 (derived from indolent BIA-ALCL), but not in the aggressive BIA-ALCL line TLBR-2. Thus, TLBR cells exhibit defective regulation of the p53 pathway in response to DNA damage, suggesting that their ability to sense DNA damage or the regulation of p53 stability may be impaired. We next examined the DNA damage sensing pathway upstream of p53 in the presence and absence of the DNA demethylating agent 5-aza-2'-deoxycytidine (AZA, 10µM for 48hrs). In all TLBR lines, ATM and ATR transcripts were expressed at much lower levels (qRT-PCR) than normal, and their expression was not significantly affected by AZA. However, compared with human T cells, CHK2 (phosphorylate P53 at Ser20) transcripts were very low in TLBR-1 and -2, but not in TLBR-3 cells. CHK2 and p21 (the main p53 target gene) transcripts after AZA were greatly increased in TLBR-2, mildly elevated in TLBR-3, and unchanged in TLBR-1 cells, suggesting that DNA methylation of the CHK2 and p21 genes may partly explain the defective p53 signaling in TLBR-2 cells. This was confirmed by detecting of CHK2 phorphrylation only in TLBR-3 cells. Mdm2, a major negative regulator of p53 protein stability, was either normal or low (qRT-PCR), and was unaffected by AZA. However, immunobloting with Mdm2 antibodies revealed increased levels of two isoforms following UV of TLBR-1 and -2, but only the small isoform was expressed in TLBR-3 cells and there was little response to UV treatment. Treatment of TLBR cells with 5 µM Nutlin-3 (Mdm2 antagonist, p53 activator, and apoptosis inducer) inhibited cell growth by 40% at day 5 (MTT assay). We conclude that these three BIA-ALCL derived cell lines share dysregulation of the p53 signaling pathway, which may contribute to the genomic instability characteristic of these BIA-ALCL cases. First two authors have equally contributed to this abstract. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Apoptosis triggered by vitexin in U937 human leukemia cells via a mitochondrial signaling pathway

2012 ◽  
Vol 28 (5) ◽  
pp. 1883-1888 ◽  
Author(s):  
CHAO-YING LEE ◽  
YUNG-SHIN CHIEN ◽  
TAI-HUI CHIU ◽  
WEN-WEN HUANG ◽  
CHI-CHENG LU ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Human Leukemia Cells ◽  
Mitochondrial Signaling

scholarly journals Exploring the Roles of HERC2 and the NEDD4L HECT E3 Ubiquitin Ligase Subfamily in p53 Signaling and the DNA Damage Response

2021 ◽  
Vol 11 ◽  
Author(s):  
Nicholas A. Mathieu ◽  
Rafael H. Levin ◽  
Donald E. Spratt
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Ubiquitin Ligase ◽  
Posttranslational Modifications ◽  
Molecular Mechanisms ◽  
P53 Signaling ◽  
Expression Of Genes ◽  
Genetic Abnormalities ◽  
Damage Response ◽  
Precise Expression

Cellular homeostasis is governed by the precise expression of genes that control the translation, localization, and termination of proteins. Oftentimes, environmental and biological factors can introduce mutations into the genetic framework of cells during their growth and division, and these genetic abnormalities can result in malignant transformations caused by protein malfunction. For example, p53 is a prominent tumor suppressor protein that is capable of undergoing more than 300 posttranslational modifications (PTMs) and is involved with controlling apoptotic signaling, transcription, and the DNA damage response (DDR). In this review, we focus on the molecular mechanisms and interactions that occur between p53, the HECT E3 ubiquitin ligases WWP1, SMURF1, HECW1 and HERC2, and other oncogenic proteins in the cell to explore how irregular HECT-p53 interactions can induce tumorigenesis.

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