scholarly journals Increased Common Fragile Site Expression, Cell Proliferation Defects, and Apoptosis following Conditional Inactivation of Mouse Hus1 in Primary Cultured Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 1044-1055 ◽  
Author(s):  
Min Zhu ◽  
Robert S. Weiss
Keyword(s):  
Cell Cycle ◽  
Cultured Cells ◽  
Chromosomal Abnormalities ◽  
Fragile Site ◽  
Loxp Site ◽  
Fragile Sites ◽  
Cell Cycle Checkpoint ◽  
Dna Breaks ◽  
Common Fragile Sites ◽  
Histone H2ax

Targeted disruption of the mouse Hus1 cell cycle checkpoint gene results in embryonic lethality and proliferative arrest in cultured cells. To investigate the essential functions of Hus1, we developed a system for the regulated inactivation of mouse Hus1 in primary fibroblasts. Inactivation of a loxP site-flanked conditional Hus1 allele by using a cre-expressing adenovirus resulted in reduced cell doubling, cell cycle alterations, and increased apoptosis. These phenotypes were associated with a significantly increased frequency of gross chromosomal abnormalities and an S-phase–specific accumulation of phosphorylated histone H2AX, an indicator of double-stranded DNA breaks. To determine whether these chromosomal abnormalities occurred randomly or at specific genomic regions, we assessed the stability of common fragile sites, chromosomal loci that are prone to breakage in cells undergoing replication stress. Hus1 was found to be essential for fragile site stability, because spontaneous chromosomal abnormalities occurred preferentially at common fragile sites upon conditional Hus1 inactivation. Although p53 levels increased after Hus1 loss, deletion of p53 failed to rescue the cell-doubling defect or increased apoptosis in conditional Hus1 knockout cells. In summary, we propose that Hus1 loss leads to chromosomal instability during DNA replication, triggering increased apoptosis and impaired proliferation through p53-independent mechanisms.

Abnormalities of 16q in Multiple Myeloma Are Associated with Poor Prognosis: 500K Gene Mapping and Expression Correlations Identify Two Potential Tumor Suppressor Genes, WWOX and CYLD.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 110-110
Author(s):  
Matthew W. Jenner ◽  
Paola E. Leone ◽  
Brian A. Walker ◽  
David C. Johnson ◽  
Laura Chiecchio ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Gene Mapping ◽  
Tumor Suppressor Genes ◽  
Poor Prognosis ◽  
Fragile Site ◽  
Fragile Sites ◽  
Common Fragile Site ◽  
Chromosome 16 ◽  
Common Fragile Sites ◽  
Potential Tumor Suppressor

Abstract Abnormalities of 16q are important recurrent events in multiple myeloma (MM). We performed FISH on CD138 selected plasma cells from 701 newly diagnosed MM patients from the LRF UKMF cytogenetics database. Gene mapping, including paired normal controls, and gene expression analysis was performed on 55 cases using the Affymetrix Human Mapping 500K Array Set and U133 Plus 2.0 Arrays respectively. 16q deletion (del16q) was identified by FISH using probes for cMAF (Abbott Diagnostics) in 131/701 cases (18.7%) and was significantly associated with deletion 17p (16.5% vs. 8.9%, p=0.006), deletion 13 (60.8% vs. 48.5%, p=0.009), deletion of IgH (22.1% vs. 11.1%, p=0.0003) and non-hyperdiploid status (58.3% vs. 42.7%, p=0.006). Del16q showed a trend to poor overall survival, mean survival 43 vs. 61 months (p=0.09), and was associated with significantly worse survival in combination with t(4;14) compared with either t(4;14) or del16q alone, mean survival 15 vs. 26 vs. 45 months respectively (p=0.006). t(14;16) was identified by FISH in 31/701 cases (4.4%) and was associated with poor prognosis, mean survival 29 vs. 54 months (p=0.005). Mapping arrays revealed loss of heterozygosity (LOH) involving all or part of 16q in 20 of 55 cases (36%) in 3 distinct patterns: uniparental disomy (UPD) of chromosome 16 or 16q in 4/55 cases (7%); deletion of chromosome 16 or the whole of 16q in 11/55 cases (20%); and interstitial deletion of small regions of 16q in 5/55 cases (10%), focused on 16q12, the location of CYLD, and 16q23, the location of WWOX. 16q LOH was distributed across translocation groups but was identified in all 4 mapping cases containing 17p deletion, supporting the association identified by FISH. As WWOX is the site of the common fragile site FRA16D and deletions at common fragile sites have been associated with DNA instability in human cancers, we assessed this using gene mapping in these 55 MM cases. Although deletions spanning other common fragile sites were identified, they were not restricted to those with 16q LOH. However, in 2 t(14;16) cases, hemizygous deletions of approximately 100kb could be identified within WWOX at the presumed translocation breakpoint. One of the t(14;16) cases had a similar hemizygous deletion within FHIT, another tumor suppressor gene located within common fragile site FRA3B, consistent with findings in other cancer types. Cases with 16q LOH or t(14;16) all had significantly reduced WWOX expression relative to cases without 16q abnormalities, confirming gene inactivation by either LOH or translocation. Cases with 16q LOH also had significantly reduced expression of two other potential tumor suppressor genes located on 16q, CYLD and RBL2. In summary, our data confirms the adverse prognosis associated with 16q translocation or deletion. Array data reveals 16q LOH occurs due to deletion or UPD with two regions involved, one defined by CYLD and the other by WWOX. WWOX is also inactivated by translocation and is associated with interstitial deletions at this and other common fragile sites. WWOX is a likely candidate gene in MM pathogenesis because of its interaction with TP53 and CYLD via its effects on NF-κB.

scholarly journals Reduced Levels of DNA Polymerase δ Induce Chromosome Fragile Site Instability in Yeast

2008 ◽  
Vol 28 (17) ◽  
pp. 5359-5368 ◽  
Author(s):  
Francene J. Lemoine ◽  
Natasha P. Degtyareva ◽  
Robert J. Kokoska ◽  
Thomas D. Petes
Keyword(s):  
Dna Polymerase ◽  
Cancer Biology ◽  
Molecular Mechanisms ◽  
Fragile Site ◽  
Fragile Sites ◽  
Dna Lesions ◽  
Catalytic Subunit ◽  
Dna Breaks ◽  
Dna Polymerase Δ ◽  
Double Strand Dna Breaks

ABSTRACT Specific regions of genomes (fragile sites) are hot spots for the chromosome rearrangements that are associated with many types of cancer cells. Understanding the molecular mechanisms regulating the stability of chromosome fragile sites, therefore, has important implications in cancer biology. We previously identified two chromosome fragile sites in Saccharomyces cerevisiae that were induced in response to the reduced expression of Pol1p, the catalytic subunit of DNA polymerase α. In the study presented here, we show that reduced levels of Pol3p, the catalytic subunit of DNA polymerase δ, induce instability at these same sites and lead to the generation of a variety of chromosomal aberrations. These findings demonstrate that a change in the stoichiometry of replicative DNA polymerases results in recombinogenic DNA lesions, presumably double-strand DNA breaks.

scholarly journals Inactivation of mouse Hus1 results in genomic instability and impaired responses to genotoxic stress

2000 ◽  
Vol 14 (15) ◽  
pp. 1886-1898 ◽  
Author(s):  
Robert S. Weiss ◽  
Tamar Enoch ◽  
Philip Leder
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Chromosomal Abnormalities ◽  
Genotoxic Stress ◽  
Eukaryotic Cell ◽  
Cell Cycle Checkpoint ◽  
Cell Cycle Checkpoints ◽  
Genome Damage

The eukaryotic cell cycle is overseen by regulatory mechanisms, termed checkpoints, that respond to DNA damage, mitotic spindle defects, and errors in the ordering of cell cycle events. The DNA replication and DNA damage cell cycle checkpoints of the fission yeastSchizosaccharomyces pombe require the hus1+(hydroxyurea sensitive) gene. To determine the role of the mouse homolog of hus1+ in murine development and cell cycle checkpoint function, we produced a targeted disruption of mouse Hus1. Inactivation of Hus1results in mid-gestational embryonic lethality due to widespread apoptosis and defective development of essential extra-embryonic tissues. DNA damage-inducible genes are up-regulated inHus1-deficient embryos, and primary cells fromHus1-null embryos contain increased spontaneous chromosomal abnormalities, suggesting that loss of Hus1 leads to an accumulation of genome damage. Embryonic fibroblasts lackingHus1 fail to proliferate in vitro, but inactivation ofp21 allows for the continued growth of Hus1-deficient cells.Hus1−/−p21−/−cells display a unique profile of significantly heightened sensitivity to hydroxyurea, a DNA replication inhibitor, and ultraviolet light, but only slightly increased sensitivity to ionizing radiation. Taken together, these results indicate that mouse Hus1 functions in the maintenance of genomic stability and additionally identify an evolutionarily-conserved role for Hus1 in mediating cellular responses to genotoxins.

scholarly journals Genome Maintenance Defects in Cultured Cells and Mice following Partial Inactivation of the Essential Cell Cycle Checkpoint Gene Hus1

2007 ◽  
Vol 27 (6) ◽  
pp. 2189-2201 ◽  
Author(s):  
Peter S. Levitt ◽  
Min Zhu ◽  
Amy Cassano ◽  
Stephanie A. Yazinski ◽  
Houchun Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stress Responses ◽  
Gene Dosage ◽  
Critical Role ◽  
Genotoxic Stress ◽  
Cell Cycle Checkpoint ◽  
Premature Senescence ◽  
Genome Maintenance ◽  
Dna Breaks ◽  
Checkpoint Gene

ABSTRACT Cell cycle checkpoints are evolutionarily conserved signaling pathways that uphold genomic integrity. Complete inactivation of the mouse checkpoint gene Hus1 results in chromosomal instability, genotoxin hypersensitivity, and embryonic lethality. To determine the functional consequences of partial Hus1 impairment, we generated an allelic series in which Hus1 expression was incrementally reduced by combining a hypomorphic Hus1 allele, Hus1 neo , with either wild-type or null (Hus1 Δ1 ) alleles. Primary Hus1 neo/Δ1 embryonic fibroblasts exhibited spontaneous chromosomal abnormalities and underwent premature senescence, while higher Hus1 expression in Hus1 neo/neo cells allowed for normal proliferation. Antioxidant treatment almost fully suppressed premature senescence in Hus1 neo/Δ1 cultures, suggesting a critical role for Hus1 in oxidative stress responses. Treatment of Hus1 neo/neo and Hus1 neo/Δ1 cells with the DNA adducting agent benzo(a)pyrene dihydrodriol epoxide resulted in a loss of cell viability that was associated with S-phase DNA damage checkpoint failure. Likewise, the DNA polymerase inhibitor aphidicolin triggered increased cell death, chromosomal aberrations, and H2AX phosphorylation, a marker for double-stranded DNA breaks, in Hus1 neo/neo and Hus1 neo/Δ1 cultures compared to controls. Despite these pronounced genome maintenance defects in cultured Hus1 neo/Δ1 and Hus1 neo/neo cells, mice of the same genotypes were born at expected frequencies and appeared grossly normal. A significant increase in micronucleus formation was observed in peripheral blood cells from Hus1neo/Δ1 mice, but reduced Hus1 expression did not cause an elevated predisposition to spontaneous tumor development or accelerate tumorigenesis in p53-deficient mice. These results identify differential effects of altered Hus1 gene dosage on genome maintenance during in vitro culture, genotoxic stress responses, embryonic development, and adult homeostasis.

Small-molecule screen reveals synergy of cell cycle checkpoint kinase inhibitors with DNA-damaging chemotherapies in medulloblastoma

2021 ◽  
Vol 13 (577) ◽  
pp. eaba7401
Author(s):  
Raelene Endersby ◽  
Jacqueline Whitehouse ◽  
Allison Pribnow ◽  
Mani Kuchibhotla ◽  
Hilary Hii ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitors ◽  
Cultured Cells ◽  
Clinical Investigation ◽  
Tumor Burden ◽  
Cell Cycle Checkpoint ◽  
Checkpoint Kinase ◽  
Cycle Checkpoint ◽  
Small Molecule Screen ◽  
Intensive Approach

Medulloblastoma (MB) consists of four core molecular subgroups with distinct clinical features and prognoses. Treatment consists of surgery, followed by radiotherapy and cytotoxic chemotherapy. Despite this intensive approach, outcome remains dismal for patients with certain subtypes of MB, namely, MYC-amplified Group 3 and TP53-mutated SHH. Using high-throughput assays, six human MB cell lines were screened against a library of 3208 unique compounds. We identified 45 effective compounds from the screen and found that cell cycle checkpoint kinase (CHK1/2) inhibition synergistically enhanced the cytotoxic activity of clinically used chemotherapeutics cyclophosphamide, cisplatin, and gemcitabine. To identify the best-in-class inhibitor, multiple CHK1/2 inhibitors were assessed in mice bearing intracranial MB. When combined with DNA-damaging chemotherapeutics, CHK1/2 inhibition reduced tumor burden and increased survival of animals with high-risk MB, across multiple different models. In total, we tested 14 different models, representing distinct MB subgroups, and data were validated in three independent laboratories. Pharmacodynamics studies confirmed central nervous system penetration. In mice, combination treatment significantly increased DNA damage and apoptosis compared to chemotherapy alone, and studies with cultured cells showed that CHK inhibition disrupted chemotherapy-induced cell cycle arrest. Our findings indicated CHK1/2 inhibition, specifically with LY2606368 (prexasertib), has strong chemosensitizing activity in MB that warrants further clinical investigation. Moreover, these data demonstrated that we developed a robust and collaborative preclinical assessment platform that can be used to identify potentially effective new therapies for clinical evaluation for pediatric MB.

scholarly journals Molecular Basis for Expression of Common and Rare Fragile Sites

2003 ◽  
Vol 23 (20) ◽  
pp. 7143-7151 ◽  
Author(s):  
Eitan Zlotorynski ◽  
Ayelet Rahat ◽  
Jennifer Skaug ◽  
Neta Ben-Porat ◽  
Efrat Ozeri ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Fragile Site ◽  
Secondary Structures ◽  
Fragile Sites ◽  
Entire Genome ◽  
Common Fragile Sites ◽  
Dna Structures ◽  
Significant Difference ◽  
Minisatellite Repeats ◽  
Genomic Regions

ABSTRACT Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.

scholarly journals ZGRF1 promotes end resection of DNA homologous recombination via forming complex with BRCA1/EXO1

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Shuang Yan ◽  
Man Song ◽  
Jie Ping ◽  
Shu-ting Lai ◽  
Xiao-yu Cao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Mammalian Cells ◽  
Cell Cycle Checkpoint ◽  
Dependent Manner ◽  
Dna Breaks ◽  
M Cell ◽  
End Resection

AbstractTo maintain genomic stability, the mammalian cells has evolved a coordinated response to DNA damage, including activation of DNA repair and cell cycle checkpoint processes. Exonuclease 1 (EXO1)-dependent excision of DNA ends is important for the initiation of homologous recombination (HR) repair of DNA breaks, which is thought to play a key role in activating the ATR-CHK1 pathway to induce G2/M cell cycle arrest. But the mechanism is still not fully understood. Here, we report that ZGRF1 forms complexes with EXO1 as well as other repair proteins and promotes DNA repair through HR. ZGRF1 is recruited to DNA damage sites in a MDC1-RNF8-BRCA1 dependent manner. Furthermore, ZGRF1 is important for the recruitment of RPA2 to DNA damage sites and the following ATR-CHK1 mediated G2/M checkpoint in response to irradiation. ZGRF1 null cells show increased sensitivity to many DNA-damaging agents, especially PARPi and irradiation. Collectively,our findings identify ZGRF1 as a novel regulator of DNA end resection and G2/M checkpoint. ZGRF1 is a potential target of radiation and PARPi cancer therapy.

scholarly journals Breakage of CRISPR/Cas9-Induced Chromosome Bridges in Mitotic Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Marina Rodriguez-Muñoz ◽  
Martina Serrat ◽  
David Soler ◽  
Anna Genescà ◽  
Teresa Anglada
Keyword(s):  
Cell Cycle ◽  
Mechanical Stress ◽  
Genome Instability ◽  
Spindle Pole ◽  
Experimental Models ◽  
Dna Breaks ◽  
Histone H2ax ◽  
Chromosome Bridge ◽  
Chromosome Bridges ◽  
Dna Ends

Chromosomal instability, the most frequent form of plasticity in cancer cells, often proceeds through the formation of chromosome bridges. Despite the importance of these bridges in tumor initiation and progression, debate remains over how and when they are resolved. In this study, we investigated the behavior and properties of chromosome bridges to gain insight into the potential mechanisms underlying bridge-induced genome instability. We report that bridges may break during mitosis or may remain unbroken until the next interphase. During mitosis, we frequently observed discontinuities in the bridging chromatin, and our results strongly suggest that a substantial fraction of chromosome bridges are broken during this stage of the cell cycle. This notion is supported by the observation that the chromatin flanking mitotic bridge discontinuities is often decorated with the phosphorylated form of the histone H2AX, a marker of DNA breaks, and by MDC1, an early mediator of the cell response to DNA breaks. Also, free 3′OH DNA ends were detected in more than half of the bridges during the final stages of cell division. However, even if detected, the DNA ends of broken bridges are not repaired in mitosis. To investigate whether mitotic bridge breakage depends on mechanical stress, we used experimental models in which chromosome bridges with defined geometry are formed. Although there was no association between spindle pole separation or the distance among non-bridge kinetochores and bridge breakage, we found a direct correlation between the distance between bridge kinetochores and bridge breakage. Altogether, we conclude that the discontinuities observed in bridges during mitosis frequently reflect a real breakage of the chromatin and that the mechanisms responsible for chromosome bridge breakage during mitosis may depend on the separation between the bridge kinetochores. Considering that previous studies identified mechanical stress or biochemical digestion as possible causes of bridge breakage in interphase cells, a multifactorial model emerges for the breakage of chromosome bridges that, according to our results, can occur at different stages of the cell cycle and can obey different mechanisms.

scholarly journals Common Fragile Site Tumor Suppressor Genes and Corresponding Mouse Models of Cancer

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Alessandra Drusco ◽  
Yuri Pekarsky ◽  
Stefan Costinean ◽  
Anna Antenucci ◽  
Laura Conti ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Mouse Models ◽  
Tumor Suppressors ◽  
Tumor Suppressor Genes ◽  
Chromosomal Abnormalities ◽  
Fragile Site ◽  
Fragile Sites ◽  
Suppressor Genes ◽  
Mouse Models Of Cancer

Chromosomal common fragile sites (CFSs) are specific mammalian genomic regions that show an increased frequency of gaps and breaks when cells are exposed to replication stressin vitro. CFSs are also consistently involved in chromosomal abnormalitiesin vivorelated to cancer. Interestingly, several CFSs contain one or more tumor suppressor genes whose structure and function are often affected by chromosomal fragility. The two most active fragile sites in the human genome are FRA3B and FRA16D where the tumor suppressor genesFHITandWWOXare located, respectively. The best approach to study tumorigenic effects of altered tumor suppressors located at CFSsin vivois to generate mouse models in which these genes are inactivated. This paper summarizes our present knowledge on mouse models of cancer generated by knocking out tumor suppressors of CFS.

scholarly journals Common fragile sites are characterised by faulty condensin loading after replication stress

2018 ◽  
Author(s):  
Lora Boteva ◽  
Ryu-Suke Nozawa ◽  
Catherine Naughton ◽  
Kumiko Samejima ◽  
William C Earnshaw ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Chromosome Instability ◽  
Replication Stress ◽  
Fragile Sites ◽  
Molecular Configuration ◽  
Common Fragile Sites ◽  
Molecular Defects ◽  
Specific Manner ◽  
Chromatin Folding

Cells coordinate interphase to mitosis transition but recurrent cytogenetic lesions appear at common fragile sites (CFSs) in a tissue-specific manner following replication stress, marking regions of instability in cancer. Despite such a distinct defect no model fully explains their molecular configuration. We show that CFSs are characterised by impaired chromatin folding manifested as disrupted mitotic structures visible using molecular FISH probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Subsequently cytogenetic and molecular lesions arise due to faulty condensin loading at CFSs, through a defect in condensin I mediated compaction and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS formation, concomitantly providing an environment for MIDAS, which, if not resolved, result in chromosome instability.

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