-
Genes Mar 2020Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells...
Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells are under replicative stress. Impairment in DNA replication, late replication timing, enrichment of A/T nucleotides that tend to form secondary structures, the paucity of active or inducible replication origins, the generation of R-loops, and the collision between replication and transcription machineries on particularly long genes are some of the reported characteristics of CFSs that may contribute to their tissue-specific fragility. Here, we validated the induction of two CFSs previously found in the human fetal lung fibroblast line, Medical Research Council strain (MRC-5), in another cell line derived from the same fetal tissue, Institute for Medical Research-90 cells (IMR-90). After induction of CFSs through aphidicolin, we confirmed the expression of the CFS 1p31.1 on chromosome 1 and CFS 3q13.3 on chromosome 3 in both fetal lines. Interestingly, these sites were found to not be fragile in lymphocytes, suggesting a role for epigenetic or transcriptional programs for this tissue specificity. Both these sites contained late-replicating genes NEGR1 (neuronal growth regulator 1) at 1p31.1 and LSAMP (limbic system-associated membrane protein) at 3q13.3, which are much longer, 0.880 and 1.4 Mb, respectively, than the average gene length. Given the established connection between long genes and CFS, we compiled information from the literature on all previously identified CFSs expressed in fibroblasts and lymphocytes in response to aphidicolin, including the size of the genes contained in each fragile region. Our comprehensive analysis confirmed that the genes found within CFSs are longer than the average human gene; interestingly, the two longest genes in the human genome are found within CFSs: Contactin Associated Protein 2 gene () in a lymphocytes' CFS, and Duchenne muscular dystrophy gene ( in a CFS expressed in both lymphocytes and fibroblasts. This indicates that the presence of very long genes is a unifying feature of all CFSs. We also obtained replication profiles of the 1p31.1 and 3q13.3 sites under both perturbed and unperturbed conditions using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence against bromodeoxyuridine (BrdU) on interphase nuclei. Our analysis of the replication dynamics of these CFSs showed that, compared to lymphocytes where these regions are non-fragile, fibroblasts display incomplete replication of the fragile alleles, even in the absence of exogenous replication stress. Our data point to the existence of intrinsic features, in addition to the presence of long genes, which affect DNA replication of the CFSs in fibroblasts, thus promoting chromosomal instability in a tissue-specific manner.
Topics: Cell Line; Cells, Cultured; Chromosome Fragile Sites; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 3; DNA Replication; Dystrophin; Female; Humans; Male; Membrane Proteins; Nerve Tissue Proteins; Organ Specificity
PubMed: 32204553
DOI: 10.3390/genes11030326 -
Cancer Research Apr 2020Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule...
Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (, and ) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.
Topics: Aphidicolin; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Colorectal Neoplasms; DNA Damage; DNA Polymerase I; DNA Polymerase II; DNA Polymerase beta; Drugs, Investigational; Enzyme Inhibitors; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Humans; Lung Neoplasms; Neoplasm Proteins; Poly-ADP-Ribose Binding Proteins; RNA, Small Interfering; Retinoids
PubMed: 32161100
DOI: 10.1158/0008-5472.CAN-19-1372 -
Nature Microbiology May 2020Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular...
Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which is formed by the repair of lesion-bearing HBV relaxed circular DNA delivered by the virions to hepatocytes. The complete and minimal set of host factors involved in cccDNA formation is unknown, largely due to the lack of a biochemical system that fully reconstitutes cccDNA formation. Here, we have developed experimental systems where various HBV relaxed-circular-DNA substrates are repaired to form cccDNA by both cell extracts and purified human proteins. Using yeast- and human-extract screenings, we identified five core components of lagging-strand synthesis as essential for cccDNA formation: proliferating cell nuclear antigen, the replication factor C complex, DNA polymerase δ, flap endonuclease 1 and DNA ligase 1. We reconstituted cccDNA formation with purified human homologues, establishing these as a minimal set of factors for cccDNA formation. We further demonstrated that treatment with the DNA-polymerase inhibitor aphidicolin diminishes cccDNA formation both in biochemical assays and in HBV-infected human cells. Together, our findings define key components in HBV cccDNA formation.
Topics: Cell Line; DNA Ligase ATP; DNA Replication; DNA, Circular; DNA, Viral; Flap Endonucleases; Genome, Viral; Hepatitis B virus; Hepatocytes; Humans; Proliferating Cell Nuclear Antigen; Replication Protein C; Virion; Virus Replication
PubMed: 32152586
DOI: 10.1038/s41564-020-0678-0 -
Environmental and Molecular Mutagenesis Oct 2020Genome instability is a hallmark of most human cancers and is exacerbated following replication stress. However, the effects that drugs/xenobiotics have in promoting...
Genome instability is a hallmark of most human cancers and is exacerbated following replication stress. However, the effects that drugs/xenobiotics have in promoting genome instability including chromosomal structural rearrangements in normal cells are not currently assessed in the genetic toxicology battery. Here, we show that drug-induced replication stress leads to increased genome instability in vitro using proliferating primary human cells as well as in vivo in rat bone marrow (BM) and duodenum (DD). p53-binding protein 1 (53BP1, biomarker of DNA damage repair) nuclear bodies were increased in a dose-dependent manner in normal proliferating human mammary epithelial fibroblasts following treatment with compounds traditionally classified as either genotoxic (hydralazine) and nongenotoxic (low-dose aphidicolin, duvelisib, idelalisib, and amiodarone). Comparatively, no increases in 53BP1 nuclear bodies were observed in nonproliferating cells. Negative control compounds (mannitol, alosteron, diclofenac, and zonisamide) not associated with cancer risk did not induce 53BP1 nuclear bodies in any cell type. Finally, we studied the in vivo genomic consequences of drug-induced replication stress in rats treated with 10 mg/kg of cyclophosphamide for up to 14 days followed by polymerase chain reaction-free whole genome sequencing (30X coverage) of BM and DD cells. Cyclophosphamide induced chromosomal structural rearrangements at an average of 90 genes, including 40 interchromosomal/intrachromosomal translocations, within 2 days of treatment. Collectively, these data demonstrate that this drug-induced genome instability test (DiGIT) can reveal potential adverse effects of drugs not otherwise informed by standard genetic toxicology testing batteries. These efforts are aligned with the food and drug administration's (FDA's) predictive toxicology roadmap initiative.
Topics: Animals; B-Lymphocytes; Biomarkers; Chromosome Aberrations; Cyclophosphamide; DNA Replication; Genome; Genomic Instability; Humans; Male; Rats; Rats, Sprague-Dawley; Whole Genome Sequencing
PubMed: 32078182
DOI: 10.1002/em.22364 -
Mutagenesis Apr 2021In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal...
In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal route. In order to explore metabolic competency and inducibility of CYP450 activating enzymes, 3D reconstructed human skin tissues were topically treated with 2-acetylaminofluorene (2-AAF) and its genotoxic metabolites, N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF) and N-hydroxy-2-aminofluorene (N-OH-2-AF), which primarily cause DNA damage by forming DNA adducts. 2-AAF did not increase DNA damage measured in the reconstructed skin micronucleus (RSMN) assay when administered in multiple applications at 24 h intervals but was detected in the skin comet assay in the presence of the DNA polymerase inhibitor aphidicolin (APC). Similarly, no increase was found with N-OH-2-AAF in the RSMN assay after multiple treatments whereas a single 3 h exposure to N-OH-2-AAF caused a large dose-related increase in the skin comet assay. A significant increase in the RSMN assay was only obtained with the highly reactive N-OH-2-AF metabolite after multiple treatments over 72 h, whereas N-OH-2-AF caused a strong increase after a single 3 h exposure in the skin comet assay. In support of these results, DNA adduct formation, measured by the 32P-postlabelling assay, was examined. Adduct levels after 2-AAF treatment for 3 h were minimal but increased >10-fold after multiple exposures over 48 h, suggesting that enzyme(s) that metabolise 2-AAF are induced in the skin models. As expected, a single 3 h exposure to N-OH-2-AAF and N-OH-2-AF resulted in adduct levels that were at least 10-fold greater than those after multiple exposures to 2-AAF despite ~100-fold lower tested concentrations. Our results demonstrate that DNA damage caused by 2-AAF metabolites is more efficiently detected in the skin comet assay than the RSMN assay and after multiple exposures and enzyme induction, 2-AAF-induced DNA damage can be detected in the APC-modified comet assay.
Topics: 2-Acetylaminofluorene; Carcinogens; DNA Adducts; DNA Damage; Fluorenes; Humans; Hydroxyacetylaminofluorene; Micronucleus Tests; Mutagens; Skin
PubMed: 31816077
DOI: 10.1093/mutage/gez044 -
Scientific Reports Nov 2019Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and - as a consequence - rates of biochemical reactions in a cell. Nanoviscosity is an...
Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and - as a consequence - rates of biochemical reactions in a cell. Nanoviscosity is an outcome of variable chemical and structural factors, which can temporarily change with cell-cycle associated changes of intracellular architecture. Thus, the question arises, whether rates of biochemical reactions depend on the point of cell cycle. In this paper we address this topic by constant observation of nanoviscosity of HeLa cells cytoplasm during S, G2 and G1 phases after Aphidicolin synchronization. For this purpose we measured diffusion rates of EGFP molecules using fluorescence correlation spectroscopy (FCS). To our surprise, a counter-intuitive stability of cytoplasmic viscosity was observed during the cell cycle. Our results hint at possible existence of robust mechanism maintaining stable physiological viscosity of the cytoplasm, despite huge structural changes during cell cycle.
Topics: Aphidicolin; Biophysical Phenomena; Cell Cycle; Cell Size; Cytoplasm; Flow Cytometry; HeLa Cells; Humans; Viscosity
PubMed: 31712575
DOI: 10.1038/s41598-019-52758-6 -
Oncogene Feb 2020Chromosomal fragile sites are genomic loci sensitive to replication stress which accumulate high levels of DNA damage, and are frequently mutated in cancers. Fragile...
Chromosomal fragile sites are genomic loci sensitive to replication stress which accumulate high levels of DNA damage, and are frequently mutated in cancers. Fragile site damage is thought to arise from the aberrant repair of spontaneous replication stress, however successful fragile site repair cannot be calculated using existing techniques. Here, we report a new assay measuring recombination-mediated repair at endogenous genomic loci by combining a sister chromatid exchange (SCE) assay with fluorescent in situ hybridization (SCE-FISH). Using SCE-FISH, we find that endogenous and exogenous replication stress generated unrepaired breaks and SCEs at fragile sites. We also find that distinct sources of replication stress induce distinct patterns of breakage: ATR inhibition induces more breaks at early replicating fragile sites (ERFS), while ERFS and late-replicating common fragile sites (CFS) are equally fragile in response to aphidicolin. Furthermore, SCEs were suppressed at fragile sites near centromeres in response to replication stress, suggesting that genomic location influences DNA repair pathway choice. SCE-FISH also measured successful recombination in human primary lymphocytes, and identificed the proto-oncogene BCL2 as a replication stress-induced fragile site. These findings demonstrate that SCE-FISH frequency at fragile sites is a sensitive indicator of replication stress, and that large-scale genome organization influences DNA repair pathway choice.
Topics: Animals; Cells, Cultured; Chromosome Fragile Sites; DNA Damage; DNA Repair; DNA Replication; DNA-Binding Proteins; Humans; In Situ Hybridization, Fluorescence; Lymphocytes; Mice; Mice, Knockout; Proto-Oncogene Mas; Recombination, Genetic; Sister Chromatid Exchange
PubMed: 31636383
DOI: 10.1038/s41388-019-1054-5 -
Cell Reports Oct 2019Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance...
Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance SUMOylation, but comprehensive insight into their substrate specificity and regulation is missing. By characterizing SENP6, we define an N-terminal multi-SIM domain as a critical determinant in targeting SENP6 to SUMO chains. Proteomic profiling reveals a network of SENP6 functions at the crossroads of chromatin organization and DNA damage response (DDR). SENP6 acts as a SUMO eraser at telomeric and centromeric chromatin domains and determines the SUMOylation status and chromatin association of the cohesin complex. Importantly, SENP6 is part of the hPSO4/PRP19 complex that drives ATR-Chk1 activation. SENP6 deficiency impairs chromatin association of the ATR cofactor ATRIP, thereby compromising the activation of Chk1 signaling in response to aphidicolin-induced replicative stress and sensitizing cells to DNA damage. We propose a general role of SENP6 in orchestrating chromatin dynamics and genome stability networks by balancing chromatin residency of protein complexes.
Topics: Amino Acid Motifs; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Checkpoint Kinase 1; Chromatin; Chromosomal Proteins, Non-Histone; Chromosomes, Human; Cysteine Endopeptidases; Genome, Human; Genomic Instability; HEK293 Cells; HeLa Cells; Humans; Nuclear Proteins; Protein Binding; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Transcription Factors; Cohesins
PubMed: 31597105
DOI: 10.1016/j.celrep.2019.08.106 -
Cell Cycle (Georgetown, Tex.) Oct 2019Chromosomal instability (CIN) causes structural and numerical chromosome aberrations and represents a hallmark of cancer. Replication stress (RS) has emerged as a driver...
Chromosomal instability (CIN) causes structural and numerical chromosome aberrations and represents a hallmark of cancer. Replication stress (RS) has emerged as a driver for structural chromosome aberrations while mitotic defects can cause whole chromosome missegregation and aneuploidy. Recently, first evidence indicated that RS can also influence chromosome segregation in cancer cells exhibiting CIN, but the underlying mechanisms remain unknown. Here, we show that chromosomally unstable cancer cells suffer from very mild RS, which allows efficient proliferation and which can be mimicked by treatment with very low concentrations of aphidicolin. Both, endogenous RS and aphidicolin-induced very mild RS cause chromosome missegregation during mitosis leading to the induction of aneuploidy. Moreover, RS triggers an increase in microtubule plus end growth rates in mitosis, an abnormality previously identified to cause chromosome missegregation in cancer cells. In fact, RS-induced chromosome missegregation is mediated by increased mitotic microtubule growth rates and is suppressed after restoration of proper microtubule growth rates and upon rescue of replication stress. Hence, very mild and cancer-relevant RS triggers aneuploidy by deregulating microtubule dynamics in mitosis.
Topics: Anaphase; Aneuploidy; Aphidicolin; Cell Line, Tumor; Cell Proliferation; Chromosomal Instability; Chromosome Segregation; DNA Damage; DNA Replication; Humans; Microtubules; Mitosis; Neoplasms
PubMed: 31448675
DOI: 10.1080/15384101.2019.1658477 -
Molecules (Basel, Switzerland) Aug 2019Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2),...
Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), along with the inhibitory effect on HSV-1 DNA polymerase, which is a key enzyme that plays an essential role in HSV-1 replication cycle. PA was found to notably inhibit HSV-1 replication (50% inhibitory concentration (IC): 1.9 μM; selectivity index (SI): 163.2) compared with the standard drug acyclovir (ACV) (IC: 2.6 μM; SI: 119.2). The combination of PA with ACV has led to potent inhibitory activity against HSV-1 replication (IC: 1.1 µM; SI: 281.8) compared with that of ACV. Moreover, PA displayed equivalent inhibitory action against HSV-2 replication (50% effective concentration (EC): 2.7 μM; SI: 114.8) compared with that of ACV (EC: 2.8 μM; SI: 110.7). The inhibition potency of PA in combination with ACV against HSV-2 replication was also detected (EC: 1.8 µM; SI: 172.2). Further, PA was observed to effectively inhibit HSV-1 DNA polymerase (as a non-nucleoside inhibitor) with respect to dTTP incorporation in a competitive inhibition mode (half maximal inhibitory concentration (IC): 0.7 μM; inhibition constant (): 0.3 μM) compared with reference drugs aphidicolin (IC: 0.8 μM; : 0.4 μM) and ACV triphosphate (ACV-TP) (IC: 0.9 μM; : 0.5 μM). It is noteworthy that the mechanism by which PA-induced anti-HSV-1 activity was related to its inhibitory action against HSV-1 DNA polymerase. Furthermore, the outcomes of in vitro experiments were authenticated using molecular docking analyses, as the molecular interactions of PA with the active sites of HSV-1 DNA polymerase and HSV-2 protease (an essential enzyme required for HSV-2 replication) were revealed. Since this is a first report on the above-mentioned properties, we can conclude that PA might be a future drug for the treatment of HSV infections as well as a promising lead molecule for further anti-HSV drug design.
Topics: Animals; Antiviral Agents; Benzoxepins; Carboxylic Acids; Chlorocebus aethiops; DNA-Directed DNA Polymerase; Herpesvirus 1, Human; Herpesvirus 2, Human; Humans; Lichens; Molecular Docking Simulation; Nucleic Acid Synthesis Inhibitors; Vero Cells; Viral Proteins; Virus Replication
PubMed: 31405197
DOI: 10.3390/molecules24162912