scholarly journals Human cells enter mitosis with damaged DNA after treatment with pharmacological concentrations of genotoxic agents

2012 ◽  
Vol 446 (3) ◽  
pp. 373-381 ◽  
Author(s):  
Philip M. Kubara ◽  
Sophie Kernéis-Golsteyn ◽  
Aurélie Studény ◽  
Brittany B. Lanser ◽  
Laurent Meijer ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Experimental System ◽  
Human Cells ◽  
Cyclin Dependent Kinase ◽  
Histone H2ax ◽  
Checkpoint Adaptation ◽  
Genotoxic Agents ◽  
G2 Phase ◽  
Damaged Dna

In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr15 Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage.

scholarly journals A comprehensive model for the proliferation–quiescence decision in response to endogenous DNA damage in human cells

2018 ◽  
Vol 115 (10) ◽  
pp. 2532-2537 ◽  
Author(s):  
Frank S. Heldt ◽  
Alexis R. Barr ◽  
Sam Cooper ◽  
Chris Bakal ◽  
Béla Novák
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Kinase Inhibitor ◽  
Human Cells ◽  
Cyclin Dependent Kinase ◽  
Restriction Point ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Serum Stimulation ◽  
G1 Cells ◽  
External Stimuli

Human cells that suffer mild DNA damage can enter a reversible state of growth arrest known as quiescence. This decision to temporarily exit the cell cycle is essential to prevent the propagation of mutations, and most cancer cells harbor defects in the underlying control system. Here we present a mechanistic mathematical model to study the proliferation–quiescence decision in nontransformed human cells. We show that two bistable switches, the restriction point (RP) and the G1/S transition, mediate this decision by integrating DNA damage and mitogen signals. In particular, our data suggest that the cyclin-dependent kinase inhibitor p21 (Cip1/Waf1), which is expressed in response to DNA damage, promotes quiescence by blocking positive feedback loops that facilitate G1 progression downstream of serum stimulation. Intriguingly, cells exploit bistability in the RP to convert graded p21 and mitogen signals into an all-or-nothing cell-cycle response. The same mechanism creates a window of opportunity where G1 cells that have passed the RP can revert to quiescence if exposed to DNA damage. We present experimental evidence that cells gradually lose this ability to revert to quiescence as they progress through G1 and that the onset of rapid p21 degradation at the G1/S transition prevents this response altogether, insulating S phase from mild, endogenous DNA damage. Thus, two bistable switches conspire in the early cell cycle to provide both sensitivity and robustness to external stimuli.

scholarly journals Telomeric Double Strand Breaks in G1 Human Cells Facilitate Formation of 5′ C-Rich Overhangs and Recruitment of TERRA

2021 ◽  
Vol 12 ◽  
Author(s):  
Christopher B. Nelson ◽  
Taghreed M. Alturki ◽  
Jared J. Luxton ◽  
Lynn E. Taylor ◽  
David G. Maranon ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Human Cells ◽  
Double Strand Breaks ◽  
End Joining ◽  
Telomeric Dna ◽  
Strand Breaks ◽  
Alternative Lengthening ◽  
Non Homologous End Joining ◽  
G1 Cells

Telomeres, repetitive nucleoprotein complexes that protect chromosomal termini and prevent them from activating inappropriate DNA damage responses (DDRs), shorten with cell division and thus with aging. Here, we characterized the human cellular response to targeted telomeric double-strand breaks (DSBs) in telomerase-positive and telomerase-independent alternative lengthening of telomere (ALT) cells, specifically in G1 phase. Telomeric DSBs in human G1 cells elicited early signatures of a DDR; however, localization of 53BP1, an important regulator of resection at broken ends, was not observed at telomeric break sites. Consistent with this finding and previously reported repression of classical non-homologous end-joining (c-NHEJ) at telomeres, evidence for c-NHEJ was also lacking. Likewise, no evidence of homologous recombination (HR)-dependent repair of telomeric DSBs in G1 was observed. Rather, and supportive of rapid truncation events, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of resected 5′ C-rich telomeric single-stranded (ss)DNA, a previously proposed marker of the recombination-dependent ALT pathway. Indeed, induction of telomeric DSBs in human ALT cells resulted in significant increases in 5′ C-rich (ss)telomeric DNA in G1, which rather than RPA, was bound by the complementary telomeric RNA, TERRA, presumably to protect these exposed ends so that they persist into S/G2 for telomerase-mediated or HR-dependent elongation, while also circumventing conventional repair pathways. Results demonstrate the remarkable adaptability of telomeres, and thus they have important implications for persistent telomeric DNA damage in normal human G1/G0 cells (e.g., lymphocytes), as well as for therapeutically relevant targets to improve treatment of ALT-positive tumors.

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.

scholarly journals SIRT1 protects the heart against doxorubicin-induced cardiotoxicity by mediating the DNA damage response via deacetylation of histone H2AX

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
A Kuno ◽  
R Hosoda ◽  
Y Horio
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Caspase 3 ◽  
Tunel Staining ◽  
H9c2 Cells ◽  
Wild Type ◽  
Histone H2ax ◽  
Funding Sources ◽  
Damage Response ◽  
Damaged Dna

Abstract Background Doxorubicin induces DNA damage not only in tumor cells but also in the cardiomyocyte, and accumulation of damaged DNA has been implicated in doxorubicin-induced cardiotoxicity. We previously found that cardiomyocyte-specific deletion of SIRT1, a NAD+-dependent histone/protein deacetylase, worsens doxorubicin-induced cardiotoxicity in mice. However, its molecular mechanism remains unclear. Phosphorylation of histone H2AX at Ser139 catalyzed by ATM (mutated in ataxia-telangiectasia) at the sites of DNA damage is a critical mediator for DNA repair. Purpose Here, we tested the hypothesis that deacetylation of H2AX by SIRT1 mediates DNA damage response to counteract doxorubicin-induced cardiotoxicity. Methods and results Wild-type (WT) mice and tamoxifen-inducible cardiomyocyte-specific SIRT1 knockout (SIRT1-cKO) mice at 3 month of age received doxorubicin (4 IP injections of 5 mg/kg/week) or a vehicle. Immunoblotting of myocardial lysates from mice 1 week after final doxorubicin showed that doxorubicin increased phospho-Ser139-H2AX level by 1.6-fold in WT, but such a response was blunted in SIRT1-cKO. Ser1981-phosphorylations of ATM induced by doxorubicin were similar in WT and SIRT1-cKO. DNA fragmentation evaluated by TUNEL staining revealed that the increase in TUNEL-positive nuclei by doxorubicin was more in SIRT1-cKO (0.13% to 0.38%) than those in WT (0.07% to 0.19%), suggesting higher DNA damage in SIRT1-cKO. In H9c2 cardiomyocytes, knockdown of SIRT1 also abolished the doxorubicin-induced Ser139-phosphorylation of H2AX without changing phospho-ATM levels. Increases in DNA damage evaluated by comet assay and cleavage of caspase-3 by doxorubicin were also enhanced in SIRT1-knockdown cells. Immunostaining for acetyl-Lys5-H2AX in the heart sections revealed that acetyl-Lys5-H2AX levels were increased in SIRT1-cKO by 58% compared with those in WT. In H9c2 cells, acetyl-Lys5-H2AX level was also increased by SIRT1 knockdown and reduced by expression of wild-type SIRT1. To test the role of the increased acetyl-Lys5-H2AX level under SIRT1 inhibition, we generated a mutant in which Lys5 was substituted to glutamine (K5Q H2AX) as a mimic of acetylated Lys5. In COS7 cells expressing WT or K5Q H2AX, Ser139-phosphorylation induced by doxorubicin was suppressed in K5Q mutant. In addition, doxorubicin-induced cleavage of caspase-3 was enhanced in H9c2 cells expressing K5Q H2AX as well as S139A H2AX, that cannot be phosphorylated at Ser139, compared with cells expressing WT H2AX. Conclusions These findings suggest that the increased Lys5 acetylation of H2AX via SIRT1 inhibition interferes Ser139 phosphorylation, leading to accumulation of damaged DNA and promotion of the apoptotic response. Such regulation of the DNA damage response contributes to protection by SIRT1 against doxorubicin-induced cardiotoxicity. FUNDunding Acknowledgement Type of funding sources: None.

scholarly journals Repression of CDK1 and Other Genes with CDE and CHR Promoter Elements during DNA Damage-Induced G2/M Arrest in Human Cells

2000 ◽  
Vol 20 (7) ◽  
pp. 2358-2366 ◽  
Author(s):  
Christophe Badie ◽  
Jane E. Itzhaki ◽  
Matthew J. Sullivan ◽  
Adam J. Carpenter ◽  
Andrew C. G. Porter
Keyword(s):  
Dna Damage ◽  
Transcriptional Repression ◽  
Human Fibroblasts ◽  
Cyclin B1 ◽  
Human Cells ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Cyclin Dependent Kinase ◽  
Promoter Regions ◽  
Topoisomerase Iiα

ABSTRACT Entry into mitosis is controlled by the cyclin-dependent kinase CDK1 and can be delayed in response to DNA damage. In some systems, such G2/M arrest has been shown to reflect the stabilization of inhibitory phosphorylation sites on CDK1. In human cells, full G2 arrest appears to involve additional mechanisms. We describe here the prolonged (>6 day) downregulation of CDK1 protein and mRNA levels following DNA damage in human cells. This silencing of gene expression is observed in primary human fibroblasts and in two cell lines with functional p53 but not in HeLa cells, where p53 is inactive. Silencing is accompanied by the accumulation of cells in G2, when CDK1 expression is normally maximal. The response is impaired by mutations in cis-acting elements (CDE and CHR) in the CDK1 promoter, indicating that silencing occurs at the transcriptional level. These elements have previously been implicated in the repression of transcription during G1that is normally lifted as cells progress into S and G2. Interestingly, we find that other genes, including those for CDC25C, cyclin A2, cyclin B1, CENP-A, and topoisomerase IIα, that are normally expressed preferentially in G2 and whose promoter regions include putative CDE and CHR elements are also downregulated in response to DNA damage. These data, together with those of other groups, support the existence of a p53-dependent, DNA damage-activated pathway leading to CHR- and CDE-mediated transcriptional repression of various G2-specific genes. This pathway may be required for sustained periods of G2 arrest following DNA damage.

scholarly journals Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8

2010 ◽  
Vol 207 (5) ◽  
pp. 973-981 ◽  
Author(s):  
Margarida Almeida Santos ◽  
Michael S.Y. Huen ◽  
Mila Jankovic ◽  
Hua-Tang Chen ◽  
Andrés J. López-Contreras ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Histone H2ax ◽  
Class Switch ◽  
Repair Defect ◽  
Dependent Pathway ◽  
Step Mechanism

53BP1 is a well-known mediator of the cellular response to DNA damage. Two alternative mechanisms have been proposed to explain 53BP1’s interaction with DNA double-strand breaks (DSBs), one by binding to methylated histones and the other via an RNF8 E3 ligase–dependent ubiquitylation pathway. The formation of RNF8 and 53BP1 irradiation-induced foci are both dependent on histone H2AX. To evaluate the contribution of the RNF8-dependent pathway to 53BP1 function, we generated RNF8 knockout mice. We report that RNF8 deficiency results in defective class switch recombination (CSR) and accumulation of unresolved immunoglobulin heavy chain–associated DSBs. The CSR DSB repair defect is milder than that observed in the absence of 53BP1 but similar to that found in H2AX−/− mice. Moreover, similar to H2AX but different from 53BP1 deficiency, RNF8−/− males are sterile, and this is associated with defective ubiquitylation of the XY chromatin. Combined loss of H2AX and RNF8 does not cause further impairment in CSR, demonstrating that the two genes function epistatically. Importantly, although 53BP1 foci formation is RNF8 dependent, its binding to chromatin is preserved in the absence of RNF8. This suggests a two-step mechanism for 53BP1 association with chromatin in which constitutive loading is dependent on interactions with methylated histones, whereas DNA damage–inducible RNF8-dependent ubiquitylation allows its accumulation at damaged chromatin.

scholarly journals DNA Damage Caused by Ionizing Radiation in Embryonic Diploid Fibroblasts WI-38 Induces Both Apoptosis and Senescence

2011 ◽  
pp. 667-677 ◽  
Author(s):  
J. CMIELOVÁ ◽  
R. HAVELEK ◽  
A. JIROUTOVÁ ◽  
R. KOHLEROVÁ ◽  
M. SEIFRTOVÁ ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ionizing Radiation ◽  
Cellular Response ◽  
Diploid Fibroblasts ◽  
Cycle Arrest ◽  
Permanent Cell ◽  
Radiation Induced ◽  
G2 Phase ◽  
Stress Induced Premature Senescence

Cellular response to ionizing radiation-induced damage depends on the cell type and the ability to repair DNA damage. Some types of cells undergo apoptosis, whereas others induce a permanent cell cycle arrest and do not proliferate. Our study demonstrates two types of response of embryonic diploid fibroblasts WI-38 to ionizing radiation. In the WI-38 cells p53 is activated, protein p21 increases, but the cells are arrested in G2 phase of cell cycle. Some of the cells die by apoptosis, but in remaining viable cells p16 increases, senescence associated DNA-damage foci occur, and senescence-associated beta-galactosidase activity increases, which indicate stress-induced premature senescence.

scholarly journals p53-dependent polyploidisation after DNA damage in G2 phase

2020 ◽  
Author(s):  
Anna Middleton ◽  
Rakesh Suman ◽  
Peter O’Toole ◽  
Karen Akopyan ◽  
Arne Lindqvist
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Tracking ◽  
Phase Cell ◽  
List Type ◽  
Dependent Manner ◽  
Cell Cycle Exit ◽  
Polyploid Cells ◽  
G2 Phase ◽  
Damaged Dna

AbstractCell cycle progression in the presence of damaged DNA can lead to accumulation of mutations and pose a risk for tumour development. In response to DNA damage in G2 phase, human cells can be forced to exit the cell cycle in a p53-p21- and APC/CCdh1-dependent manner. Cells that exit the cell cycle in G2 phase become senescent, but it is unclear what determines this commitment and whether other cell fates occur. We find that a subset of immortalised RPE-1 cells and primary human fibroblasts spontaneously initiate DNA re-replication several days after forced cell cycle exit in G2 phase. By combining single cell tracking for more than a week with quantitative immunofluorescence, we find that the resulting polyploid cells contain increased levels of damaged DNA and frequently exit the cell cycle again in the next G2 phase. Subsequently, these cells either enter senescence or commit to another round of DNA re-replication, further increasing the ploidy. At least a subset of the polyploid cells show abnormal centrosome numbers or localisation. In conclusion, cells that are forced to exit the cell cycle in G2 phase face multiple choices that lead to various phenotypes, including propagation of cells with different ploidies. Our findings suggest a mechanism by which p53-positive cells can evade senescence that risks genome integrity.Main points-Cell cycle exit from G2 phase does not necessarily lead to senescence-Resumption of proliferation after G2 phase cell cycle exit starts with DNA replication-Successive cell cycle exits lead to propagation of cells with different ploidies-A p53-dependent mechanism allows eventual proliferation after DNA damage

scholarly journals An ATM/Wip1-dependent timer controls the minimal duration of a DNA-damage mediated cell cycle arrest

2016 ◽  
Author(s):  
Himjyot Jaiswal ◽  
Jan Benada ◽  
Erik Mullers ◽  
Karen Akopyan ◽  
Kamila Burdova ◽  
...  
Keyword(s):  
Experimental Data ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Dna Lesions ◽  
Cycle Arrest ◽  
Checkpoint Adaptation ◽  
Global Spread ◽  
A Cell ◽  
G2 Phase

After DNA damage, the cell cycle is arrested to avoid propagation of mutations. In G2 phase, the arrest is initiated by ATM/ATR-dependent signalling that blocks mitosis-promoting kinases as Plk1. Interestingly, Plk1 can counteract ATR-dependent signalling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of targets including Kap1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing a cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modelling we propose that the minimal duration of a cell cycle arrest is controlled by a timer. Our model shows how cell cycle re-start can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.

scholarly journals Role for Lyn Tyrosine Kinase as a Regulator of Stress-Activated Protein Kinase Activity in Response to DNA Damage

2000 ◽  
Vol 20 (15) ◽  
pp. 5370-5380 ◽  
Author(s):  
Kiyotsugu Yoshida ◽  
Ralph Weichselbaum ◽  
Surender Kharbanda ◽  
Donald Kufe
Keyword(s):  
Dna Damage ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Cellular Response ◽  
Protein Tyrosine Kinase ◽  
Genotoxic Stress ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Activity ◽  
Genotoxic Agents

ABSTRACT The cellular response to DNA damage includes activation of the nuclear Lyn protein tyrosine kinase. Using cells deficient in Lyn expression, the present studies demonstrate that Lyn is required in part for induction of the stress-activated protein kinase (SAPK) in the response to 1-β-d-arabinofuranosylcytosine (ara-C) and other genotoxic agents. By contrast, exposure of Lyn-deficient cells to ara-C, ionizing radiation, or cisplatin had no effect on activation of extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase. Similar findings were obtained in cells stably expressing a kinase-inactive, dominant-negative Lyn(K-R) mutant. Coexpression studies demonstrate that Lyn, but not Lyn(K-R), induces SAPK activity. In addition, the results demonstrate that Lyn activates SAPK by an MKK7-dependent, SEK1-independent mechanism. As MEKK1 functions upstream to MKK7 and SAPK, the finding that a dominant-negative MEKK1(K-M) mutant blocks Lyn-induced SAPK activity supports involvement of the MEKK1→MKK7 pathway. The results also demonstrate that inhibition of Lyn-induced SAPK activity abrogates the apoptotic response of cells to genotoxic stress. These findings indicate that activation of SAPK by DNA damage is mediated in part by Lyn and that the Lyn→MEKK1→MKK7→SAPK pathway is functional in the induction of apoptosis by genotoxic agents.

Sign in / Sign up

Export Citation Format

Share Document