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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 -
BMC Genomics Jul 2019Replication stress (RS) gives rise to DNA damage that threatens genome stability. RS can originate from different sources that stall replication by diverse mechanisms....
BACKGROUND
Replication stress (RS) gives rise to DNA damage that threatens genome stability. RS can originate from different sources that stall replication by diverse mechanisms. However, the mechanism underlying how different types of RS contribute to genome instability is unclear, in part due to the poor understanding of the distribution and characteristics of damage sites induced by different RS mechanisms.
RESULTS
We use ChIP-seq to map γH2AX binding sites genome-wide caused by aphidicolin (APH), hydroxyurea (HU), and methyl methanesulfonate (MMS) treatments in human lymphocyte cells. Mapping of γH2AX ChIP-seq reveals that APH, HU, and MMS treatments induce non-random γH2AX chromatin binding at discrete regions, suggesting that there are γH2AX binding hotspots in the genome. Characterization of the distribution and sequence/epigenetic features of γH2AX binding sites reveals that the three treatments induce γH2AX binding at largely non-overlapping regions, suggesting that RS may cause damage at specific genomic loci in a manner dependent on the fork stalling mechanism. Nonetheless, γH2AX binding sites induced by the three treatments share common features including compact chromatin, coinciding with larger-than-average genes, and depletion of CpG islands and transcription start sites. Moreover, we observe significant enrichment of SINEs in γH2AX sites in all treatments, indicating that SINEs may be a common barrier for replication polymerases.
CONCLUSIONS
Our results identify the location and common features of genome instability hotspots induced by different types of RS, and help in deciphering the mechanisms underlying RS-induced genetic diseases and carcinogenesis.
Topics: Aphidicolin; Binding Sites; Cell Line; Chromosome Mapping; DNA Replication; Genome, Human; Genomic Instability; Histones; Humans; Hydroxyurea; Stress, Physiological; Sulfinic Acids
PubMed: 31299901
DOI: 10.1186/s12864-019-5934-4 -
Molecular Cancer Research : MCR Aug 2019Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress....
Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress. This unusual form of DNA synthesis occurs in the absence of RAD51 or BRCA2, which led to the identification of RAD52 as a key player in this process. Notably, mitotic DNA synthesis is predominantly observed at chromosome loci that colocalize with FANCD2 foci. However, the role of this protein in mitotic DNA synthesis remains largely unknown. In this study, we investigated the role of FANCD2 and its interplay with RAD52 in mitotic DNA synthesis using aphidicolin as a universal inducer of this process. After examining eight human cell lines, we provide evidence for FANCD2 rather than RAD52 as a fundamental supporter of mitotic DNA synthesis. In cancer cell lines, FANCD2 exerts this role independently of RAD52. Surprisingly, RAD52 is dispensable for mitotic DNA synthesis in noncancerous cell lines, but these cells strongly depend on FANCD2 for this process. Therefore, RAD52 functions selectively in cancer cells as a secondary regulator in addition to FANCD2 to facilitate mitotic DNA synthesis. As an alternative to aphidicolin, we found partial inhibition of origin licensing as an effective way to induce mitotic DNA synthesis preferentially in cancer cells. Importantly, cancer cells still perform mitotic DNA synthesis by dual regulation of FANCD2 and RAD52 under such conditions. IMPLICATIONS: These key differences in mitotic DNA synthesis between cancer and noncancerous cells advance our understanding of this mechanism and can be exploited for cancer therapies.
Topics: Cell Nucleus; Cells, Cultured; DNA; DNA Damage; DNA Repair; Fanconi Anemia Complementation Group D2 Protein; Fibroblasts; Humans; Mitosis; Neoplasms; Rad52 DNA Repair and Recombination Protein; Retinal Pigment Epithelium
PubMed: 31113828
DOI: 10.1158/1541-7786.MCR-19-0057 -
Cartilage Oct 2021Although tissue engineering is a promising option for articular cartilage repair, it has been challenging to generate functional cartilaginous tissue. While the...
OBJECTIVE
Although tissue engineering is a promising option for articular cartilage repair, it has been challenging to generate functional cartilaginous tissue. While the synthetic response of chondrocytes can be influenced by various means, most approaches treat chondrocytes as a homogeneous population that would respond similarly. However, isolated cells heterogeneously progress through the cell cycle, which can affect macromolecular biosynthesis. As it is possible to synchronize cells within discrete cell cycle phases, the purpose of this study was to investigate the effects of cell cycle synchronization on the chondrogenic potential of primary articular chondrocytes.
DESIGN
Different methods of cell synchronization (serum starvation, thymidine, nocodazole, aphidicolin, and RO-3306) were tested for their ability to synchronize primary articular chondrocytes during the process of cell isolation. Cells (unsynchronized and synchronized) were then encapsulated in alginate gels, cultured for 4 weeks, and analyzed for their structural and biochemical properties.
RESULTS
The double-thymidine method yielded the highest level of cell purity, with cells synchronized in S phase. While the cells started to lose synchronization after 24 hours, tissue constructs developed from initially S phase synchronized cells had significantly higher glycosaminoglycan and collagen II amounts than those developed using unsynchronized cells.
CONCLUSIONS
Initial synchronization led to long-term changes in cartilaginous tissue formation. This effect was postulated to be due to the rapid auto-induction of TGF-βs by actively dividing S phase cells, thereby stimulating chondrogenesis. Cell synchronization methods may also be applied in conjunction with redifferentiation methods to improve the chondrogenic potential of dedifferentiated or diseased chondrocytes.
Topics: Cell Cycle; Cells, Cultured; Chondrocytes; Chondrogenesis; Thymidine
PubMed: 30971093
DOI: 10.1177/1947603519841677