scholarly journals Loss of p53 suppresses replication-stress-induced DNA breakage in G1/S checkpoint deficient cells

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Bente Benedict ◽  
Tanja van Harn ◽  
Marleen Dekker ◽  
Simone Hermsen ◽  
Asli Kucukosmanoglu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Replication Stress ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Origin Firing ◽  
Strand Breaks ◽  
Dna Breakage ◽  
Cycle Arrest

In cancer cells, loss of G1/S control is often accompanied by p53 pathway inactivation, the latter usually rationalized as a necessity for suppressing cell cycle arrest and apoptosis. However, we found an unanticipated effect of p53 loss in mouse and human G1-checkpoint-deficient cells: reduction of DNA damage. We show that abrogation of the G1/S-checkpoint allowed cells to enter S-phase under growth-restricting conditions at the expense of severe replication stress manifesting as decelerated DNA replication, reduced origin firing and accumulation of DNA double-strand breaks. In this system, loss of p53 allowed mitogen-independent proliferation, not by suppressing apoptosis, but rather by restoring origin firing and reducing DNA breakage. Loss of G1/S control also caused DNA damage and activation of p53 in an in vivo retinoblastoma model. Moreover, in a teratoma model, loss of p53 reduced DNA breakage. Thus, loss of p53 may promote growth of incipient cancer cells by reducing replication-stress-induced DNA damage.

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.

scholarly journals RepID-deficient cancer cells are sensitized to a drug targeting p97/VCP segregase

2021 ◽  
Author(s):  
Sang-Min Jang ◽  
Christophe E. Redon ◽  
Haiqing Fu ◽  
Fred E. Indig ◽  
Mirit I. Aladjem
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Cell Sorting ◽  
Clonogenic Survival ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Ubiquitinated Proteins ◽  
Damage Response ◽  
Survival Assay

Abstract Background The p97/valosin-containing protein (VCP) complex is a crucial factor for the segregation of ubiquitinated proteins in the DNA damage response and repair pathway. Objective We investigated whether blocking the p97/VCP function can inhibit the proliferation of RepID-deficient cancer cells using immunofluorescence, clonogenic survival assay, fluorescence-activated cell sorting, and immunoblotting. Result p97/VCP was recruited to chromatin and colocalized with DNA double-strand breaks in RepID-deficient cancer cells that undergo spontaneous DNA damage. Inhibition of p97/VCP induced death of RepID-depleted cancer cells. This study highlights the potential of targeting p97/VCP complex as an anticancer therapeutic approach. Conclusion Our results show that RepID is required to prevent excessive DNA damage at the endogenous levels. Localization of p97/VCP to DSB sites was induced based on spontaneous DNA damage in RepID-depleted cancer cells. Anticancer drugs targeting p97/VCP may be highly potent in RepID-deficient cells. Therefore, we suggest that p97/VCP inhibitors synergize with RepID depletion to kill cancer cells.

scholarly journals Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

2019 ◽  
Vol 116 (39) ◽  
pp. 19552-19562 ◽  
Author(s):  
Justine Sitz ◽  
Sophie Anne Blanchet ◽  
Steven F. Gameiro ◽  
Elise Biquand ◽  
Tia M. Morgan ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Genomic Instability ◽  
E3 Ligase ◽  
Human Papillomaviruses ◽  
Double Strand Breaks ◽  
Nuclear Bodies ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell’s DNA replication and repair machineries to replicate their own genomes. How this host–pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

scholarly journals Focused ultrasound radiosensitizes human cancer cells by enhancement of DNA damage

2021 ◽  
Author(s):  
Xinrui Zhang ◽  
Mariana Bobeica ◽  
Michael Unger ◽  
Anastasia Bednarz ◽  
Bjoern Gerold ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Metabolic Activity ◽  
Focused Ultrasound ◽  
Double Strand Breaks ◽  
High Intensity Focused Ultrasound ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Abstract Purpose High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40–47 °C (thermal dose CEM43 ≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. Methods An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467 MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose X‑ray irradiation (10 Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST‑1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). Results The FUS intensities of 213 (1.147 MHz) and 225 W/cm2 (1.467 MHz) induced HT for 30 min at mean temperatures of 45.20 ± 2.29 °C (CEM43 = 436 ± 88) and 45.59 ± 1.65 °C (CEM43 = 447 ± 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48 h (RT: 96.47 ± 8.29%; FUS+RT: 79.38 ± 14.93%; p = 0.012) and in PC-3 cells 72 h (54.20 ± 10.85%; 41.01 ± 11.17%; p = 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. Conclusion Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage.

scholarly journals βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1

2019 ◽  
Vol 27 (4) ◽  
pp. 1200-1213 ◽  
Author(s):  
Ainhoa Nieto ◽  
Makoto R. Hara ◽  
Victor Quereda ◽  
Wayne Grant ◽  
Vanessa Saunders ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Molecular Scaffold ◽  
Strand Breaks

Abstract Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the β2-adrenergic-βarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, βarrestin-1 (βarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether βarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice βarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that βarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of βarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, βarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, βarr1 is an important regulator of double strand break repair, and disruption of the βarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

scholarly journals Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks

2012 ◽  
Vol 40 (12) ◽  
pp. 5794-5794
Author(s):  
C. Lundin ◽  
M. North ◽  
K. Erixon ◽  
K. Walters ◽  
D. Jenssen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Methyl Methanesulfonate ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Heat Labile

scholarly journals DNA-dependent macromolecular condensation drives self-assembly of the meiotic DNA break machinery

2020 ◽  
Author(s):  
Corentin Claeys Bouuaert ◽  
Stephen Pu ◽  
Juncheng Wang ◽  
Dinshaw J. Patel ◽  
Scott Keeney
Keyword(s):  
Self Assembly ◽  
Chromosome Structure ◽  
Coiled Coil ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Breakage ◽  
And Control

Formation of meiotic DNA double-strand breaks (DSBs) by Spo11 is tightly regulated and tied to chromosome structure, but the higher-order assemblies that execute and control DNA breakage are poorly understood. We address this question through molecular characterization of Saccharomyces cerevisiae RMM proteins (Rec114, Mei4 and Mer2)—essential, conserved components of the DSB machinery. Each subcomplex of Rec114–Mei4 (2:1 heterotrimer) or Mer2 (homotetrameric coiled coil) is monodisperse in solution, but they independently condense with DNA into dynamic, reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive condensation, which correlates with DSB formation in vivo. Condensates fuse into mixed Rec114–Mei4–Mer2 clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centers of DSB activity. We propose that multilayered control of Spo11 arises from recruitment of regulatory components and modulation of biophysical properties of the condensates.

scholarly journals DNA Damage-Induced Cell Cycle Regulation and Function of Novel Chk2 Phosphoresidues

2006 ◽  
Vol 26 (21) ◽  
pp. 7832-7845 ◽  
Author(s):  
Giacomo Buscemi ◽  
Luigi Carlessi ◽  
Laura Zannini ◽  
Sofia Lisanti ◽  
Enrico Fontanella ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Dna Double Strand Breaks ◽  
Independent Manner ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
And Function ◽  
Cycle Regulation

ABSTRACT Chk2 kinase is activated by DNA damage to regulate cell cycle arrest, DNA repair, and apoptosis. Phosphorylation of Chk2 in vivo by ataxia telangiectasia-mutated (ATM) on threonine 68 (T68) initiates a phosphorylation cascade that promotes the full activity of Chk2. We identified three serine residues (S19, S33, and S35) on Chk2 that became phosphorylated in vivo rapidly and exclusively in response to ionizing radiation (IR)-induced DNA double-strand breaks in an ATM- and Nbs1-dependent but ataxia telangiectasia- and Rad3-related-independent manner. Phosphorylation of these residues, restricted to the G1 phase of the cell cycle, was induced by a higher dose of IR (>1 Gy) than that required for phosphorylation of T68 (0.25 Gy) and declined by 45 to 90 min, concomitant with a rise in Chk2 autophosphorylation. Compared to the wild-type form, Chk2 with alanine substitutions at S19, S33, and S35 (Chk2S3A) showed impaired dimerization, defective auto- and trans-phosphorylation activities, and reduced ability to promote degradation of Hdmx, a phosphorylation target of Chk2 and regulator of p53 activity. Besides, Chk2S3A failed to inhibit cell growth and, in response to IR, to arrest G1/S progression. These findings underscore the critical roles of S19, S33, and S35 and argue that these phosphoresidues may serve to fine-tune the ATM-dependent response of Chk2 to increasing amounts of DNA damage.

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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