Neuronal Cell Cycle Re-Entry Enhances Neuropathological Features in App NLF Knock-In Mice

Background: Aberrant cell cycle re-entry is a well-documented process occurring early in Alzheimer’s disease (AD). This is an early feature of the disease and may contribute to disease pathogenesis. Objective: To assess the effect of forced neuronal cell cycle re-entry in mice expressing humanized Aβ, we crossed our neuronal cell cycle re-entry mouse model with App NLF knock-in (KI) mice. Methods: Our neuronal cell cycle re-entry (NCCR) mouse model is bitransgenic mice heterozygous for both Camk2a-tTA and TRE-SV40T. The NCCR mice were crossed with App NLF KI mice to generate NCCR-App NLF animals. Using this tet-off system, we triggered NCCR in our animals via neuronal expression of SV40T starting at 1 month of age. The animals were examined at the following time points: 9, 12, and 18 months of age. Various neuropathological features in our mice were evaluated by image analysis and stereology on brain sections stained using either immunofluorescence or immunohistochemistry. Results: We show that neuronal cell cycle re-entry in humanized Aβ plaque producing App NLF KI mice results in the development of additional AD-related pathologies, namely, pathological tau, neuroinflammation, brain leukocyte infiltration, DNA damage response, and neurodegeneration. Conclusion: Our findings show that neuronal cell cycle re-entry enhances AD-related neuropathological features in App NLF mice and highlight our unique AD mouse model for studying the pathogenic role of aberrant cell cycle re-entry in AD.

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Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgz185 ◽

2019 ◽

Vol 105 (3) ◽

pp. 839-853

Author(s):

Aglaia Kyrilli ◽

David Gacquer ◽

Vincent Detours ◽

Anne Lefort ◽

Frédéric Libert ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

Iodide Uptake ◽

Transcriptomic Response ◽

Uptake Inhibition ◽

Γ Radiation ◽

Damage Response

Abstract Background The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Methods Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Results Overall, the thyrocyte responses following exposure to β or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. Conclusions TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

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Involvement of Death-Associated Protein Kinases In DNA Damage Responses of B-ALL Cells.

Blood ◽

10.1182/blood.v116.21.3369.3369 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3369-3369

Author(s):

Magali Humbert ◽

Michaela Medova ◽

Barbara Geering ◽

Wieslawa Blank-Liss ◽

Hans-Uwe Simon ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Gamma Irradiation ◽

Protein Kinases ◽

Dna Damage Response ◽

Lymphoblastic Leukemia ◽

Damage Response ◽

Dna Damage Responses

Abstract Abstract 3369 Intact DNA damage response pathways are important for genomic fidelity of cells in order to avoid tumor formation. On the other hand, inhibition of DNA repair provides an important mechanism to enhance the therapeutic efficacy of DNA damaging agents such as gamma-irradiation. Thus, it is important to identify novel players in DNA damage response that might represent novel targets for combination therapies. Death-associated protein kinases (DAPK) are serine/threonine kinases believed to be involved in cell death and autophagy mechanisms, whereby particularly the role of DAPK1 has previously been investigated. The DAPK family is composed of five members: DAPK1, DAPK2 (or DRP-1), DAPK3 (or ZIP kinase), DRAK1 and DRAK2. DAPK1 and DAPK2 share 80% homology in the catalytic domain. Generally, the role of DAPK in DNA damage responses is not well studied. To analyze the role of DAPK1 and DAPK2 in response to gamma-irradiation, we used p53 wild-type REH B-cell acute lymphoblastic leukemia (B-ALL) cells as a model. In response to irradiation, DAPK1 protein expression increased paralleled by an increased of total p53, phospho-Ser20-p53 and p21WAF1/CIP1. DAPK2 expression, however, did not increase. Since upregulation of p21WAF1/CIP1, a classical p53 target in response to DNA damage leads to cell cycle arrest, we asked whether knocking down DAPK1 or DAPK2 might affect the cell cycle. Interestingly, knocking down DAPK2 but not DAPK1 led to a significant increase of S-phase cells upon irradiation. Moreover, knocking down DAPK2 attenuated the induction of DAPK1 upon irradiation indicating a DAPK2-DAPK1 cascade in DNA damage responses. Next, given the significant role of p21WAF1/CIP1 and p53 in DNA damage responses, we tested if DAPK2 might directly participate in a novel signaling pathway by interacting with these proteins. Indeed, pull down assays revealed that p21WAF1/CIP1 and p53 are novel DAPK2 interacting proteins. Clearly, further experiments are needed to define the DAPK2-DAPK1-p53- p21WAF1/CIP1 network in DNA repair pathways. In conclusion, we identified a novel role for DAPK1 and DAPK2 in DNA damage responses of B-ALL cells and propose a novel DAPK2/DAPK1/p53/ p21WAF1/CIP1 DNA damage regulatory pathway. Disclosures: No relevant conflicts of interest to declare.

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SET8 Is a Potential Therapeutic Target in MM

Blood ◽

10.1182/blood.v128.22.4435.4435 ◽

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.

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A Role for NIPA in DNA Damage Response.

Blood ◽

10.1182/blood.v104.11.1265.1265 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1265-1265

Author(s):

Christine von Klitzing ◽

Florian Bassermann ◽

Stephan W. Morris ◽

Christian Peschel ◽

Justus Duyster

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

Phosphorylation Site ◽

Genotoxic Stress ◽

S Phase ◽

Damage Response ◽

A Cell ◽

Cycle Dependent

Abstract The nuclear interaction partner of ALK (NIPA) is a nuclear protein identified by our group in a screen for NPM-ALK interaction partners. We recently reported that NIPA is an F-box protein that assembles with SKP1, Cul1 and Roc1 to establish a novel SCF-type E3 ubiquitin ligase. The formation of the SCFNIPA complex is regulated by cell cycle-dependent phosphorylation of NIPA that restricts SCFNIPA assembly from G1- to late S-phase, thus allowing its substrates to be active from late S-phase throughout mitosis. Proteins involved in cell cycle regulation frequently play a role in DNA damage checkpoints. We therefore sought to determine whether NIPA has a function in the cellular response to genotoxic stress. For this reason we treated NIH/3T3 cells with various DNA-damaging agents. Surprisingly, we observed phosphorylation of NIPA in response to some of these agents, including UV radiation. This phosphorylation was cell cycle phase independent and thus independent of the physiological cell cycle dependent phosphorylation of NIPA. The relevant phosphorylation site is identical to the respective site in the course of cell cycle-dependent phosphorylation of NIPA. Thus, phosphorylation of NIPA upon genotoxic stress would inactivate the SCFNIPA complex in a cell cycle independent manner. Interestingly, this phosphorylation site lies within a consensus site of the Chk1/Chk2 checkpoint kinases. These kinases are central to DNA damage checkpoint signaling. Chk1 is activated by ATR in response to blocked replication forks as they occur after treatment with UV. We performed experiments using the ATM/ATR inhibitor caffeine and the Chk1 inhibitor SB218078 to investigate a potential role of Chk1 in NIPA phosphorylation. Indeed, we found both inhibitors to prevent UV-induced phosphorylation of NIPA. Current experiments applying Chk1 knock-out cells will unravel the role of Chk1 in NIPA phosphorylation. Additional experiments were performed to investigate a function for NIPA in DNA-damage induced apoptosis. In this regard, we observed overexpression of NIPA WT to induce apoptosis in response to UV, whereas no proapoptotic effect was seen with the phosphorylation deficient NIPA mutant. Therefore, the phosphorylated form of NIPA may be involved in apoptotic signaling pathways. In summary, we present data suggesting a cell cycle independent function for NIPA. This activity is involved in DNA damage response and may be involved in regulating apoptosis upon genotoxic stress.

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c-Abl-mediated tyrosine phosphorylation of JunB is required for Adriamycin-induced expression of p21

Biochemical Journal ◽

10.1042/bj20150372 ◽

2015 ◽

Vol 471 (1) ◽

pp. 67-77 ◽

Cited By ~ 8

Author(s):

Noritaka Yamaguchi ◽

Ryuzaburo Yuki ◽

Sho Kubota ◽

Kazumasa Aoyama ◽

Takahisa Kuga ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Tyrosine Phosphorylation ◽

Dna Damage Response ◽

Induced Expression ◽

Cell Cycle Inhibitor ◽

Damage Response

We analysed the role of c-Abl-mediated tyrosine phosphorylation of JunB in Adriamycin-induced DNA damage response. Tyrosine phosphorylation of JunB was found to be required for expression of the cell cycle inhibitor p21 upon Adriamycin stimulation.

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Role of the Cell Cycle Re-Initiation in DNA Damage Response of Post-Mitotic Cells and Its Implication in the Pathogenesis of Neurodegenerative Diseases

Rejuvenation Research ◽

10.1089/rej.2015.1717 ◽

2016 ◽

Vol 19 (2) ◽

pp. 131-139 ◽

Cited By ~ 11

Author(s):

Paulina Tokarz ◽

Kai Kaarniranta ◽

Janusz Blasiak

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Neurodegenerative Diseases ◽

Dna Damage Response ◽

Damage Response ◽

Mitotic Cells

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The Role of UBR5 Mutations in the Pathogenesis of Mantle CELL Lymphoma

Blood ◽

10.1182/blood.v128.22.4124.4124 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4124-4124

Author(s):

Olga Kutovaya ◽

Stacy Hung ◽

Hughes Christopher ◽

Randy D Gascoyne ◽

Morin Gregg ◽

...

Keyword(s):

Mass Spectrometry ◽

Cell Cycle ◽

Dna Damage ◽

Mantle Cell Lymphoma ◽

Dna Damage Response ◽

Cell Lymphoma ◽

Interacting Proteins ◽

Mantle Cell ◽

Damage Response

Abstract Intro: Mantle cell lymphoma (MCL) accounts for 6% of non-Hodgkin lymphomas and represents a particularly challenging disease with patient outcomes inferior to most other lymphoma subtypes. Using targeted capture sequencing of MCL biopsy samples, we recently reported frequent mutations (18%) in UBR5, a gene encoding an E3 ubiquitin-protein ligase that has not been previously implicated in lymphomagenesis. All mutations were clustered within 100bp in or around exon 58 of UBR5 and truncate the reading frame or change a key lysine residue. These mutations are predicted to result in the loss of the conserved cysteine residue in the HECT-domain, which is responsible for binding the ubiquitin co-factor. The recurrence and clustering of UBR5 mutations suggest their critical pathogenic involvement in a subgroup of MCL that might be therapeutically targetable. The aim of this study is to determine UBR5 mutation-associated proteome changes and altered cell signaling. Methods: As seen in MCL patients, mutations in exon 58 of UBR5 were introduced to three MCL cell lines (Granta-519, Jeko-1, and Mino) using the CRISPR-Cas9 genome engineering tool. First, mass spectrometry-based immunoprecipitation proteomics (IP-MS) was employed to identify differences in UBR5 interacting partners between UBR5 mutant and wildtype (WT) cells. Candidate UBR5 interacting proteins were validated by flow cytometry, western blotting, co-immunoprecipitation, and immunofluorescence. Next, global proteomes of UBR5 mutants and WT were analyzed by Tandem Mass Tag (TMT)-based mass spectrometry quantification to identify proteins with differential expression due to the UBR5 mutations. Results: The IP-MS analysis of WT vs UBR5 mutants revealed histone and cell cycle control proteins as candidate differential UBR5 interacting proteins (p<0.05). Particularly, histones H1, H4, and H2AFX, as well as the cell cycle genes CDC5L, BUB3, MAP4, RAD50 and CDK11B were identified as candidate UBR5 interacting partners. The global proteome analysis identified a set of differentially expressed genes (mutant vs wt; p<0.05) that are common among the MCL cell lines with the same direction of change. Gene ontology analysis of this set revealed DNA damage response, chromosome organization, and cell cycle response pathways as the predominant pathways affected. Moreover, our preliminary functional studies indicate constitutive phosphorylation of H2AFX in UBR5 mutants vs WT in line with the role of UBR5 in DNA damage response. Conclusions: Our results are consistent with UBR5 functioning as a key regulator of cell signalling and strongly suggest UBR5 as a novel regulator of histone modifications and DNA damage response. These findings provide an experimentally valid platform for further functional investigation and testing of target therapies for MCL harbouring UBR5 mutations. Disclosures No relevant conflicts of interest to declare.

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