Comet Tail Formation Research Articles

Protective Effect of 3,4-Dihydroxybenzoic Acid Isolated from Cladophora wrightiana Harvey Against Ultraviolet B Radiation-Induced Cell Damage in Human HaCaT Keratinocytes

The aim of the present study was to elucidate the protective properties of 3,4-dihydroxybenzoic acid (DBA) isolated from Cladophora wrightiana Harvey (a green alga) against ultraviolet B (UVB)-induced damage to human HaCaT keratinocytes. DBA exhibited scavenging actions against the 1,1-diphenyl-2-picrylhydrazyl radical, the superoxide anion, and the hydroxyl radical. Furthermore, DBA decreased the levels of intracellular reactive oxygen species generated by hydrogen peroxide or UVB treatment of the cells. DBA also decreased the UVB-augmented levels of phospho-histone H2A.X and the extent of comet tail formation, which are both indications of DNA damage. In addition, the compound safeguarded keratinocytes from UVB-induced injury by reversing the production of apoptotic bodies, overturning the disruption of mitochondrial membrane potential, increasing the expression of the anti-apoptotic protein, B-cell lymphoma 2, and decreasing the expression of the pro-apoptotic proteins, Bcl-2-associated X and cleaved caspase-3. Taken together, these results demonstrate that DBA isolated from a green alga protects human keratinocytes against UVB-induced oxidative stress and apoptosis.

Fucoxanthin Protects Cultured Human Keratinocytes against Oxidative Stress by Blocking Free Radicals and Inhibiting Apoptosis

Fucoxanthin is an important carotenoid derived from edible brown seaweeds and is used in indigenous herbal medicines. The aim of the present study was to examine the cytoprotective effects of fucoxanthin against hydrogen peroxide-induced cell damage. Fucoxanthin decreased the level of intracellular reactive oxygen species, as assessed by fluorescence spectrometry performed after staining cultured human HaCaT keratinocytes with 2',7'-dichlorodihydrofl uorescein diacetate. In addition, electron spin resonance spectrometry showed that fucoxanthin scavenged hydroxyl radical generated by the Fenton reaction in a cell-free system. Fucoxanthin also inhibited comet tail formation and phospho-histone H2A.X expression, suggesting that it prevents hydrogen peroxideinduced cellular DNA damage. Furthermore, the compound reduced the number of apoptotic bodies stained with Hoechst 33342, indicating that it protected keratinocytes against hydrogen peroxide-induced apoptotic cell death. Finally, fucoxanthin prevented the loss of mitochondrial membrane potential. These protective actions were accompanied by the down-regulation of apoptosispromoting mediators (i.e., B-cell lymphoma-2-associated x protein, caspase-9, and caspase-3) and the up-regulation of an apoptosis inhibitor (B-cell lymphoma-2). Taken together, the results of this study suggest that fucoxanthin defends keratinocytes against oxidative damage by scavenging ROS and inhibiting apoptosis.

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G2/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.

Sterigmatocystin-Induced DNA Damage Triggers G2 Arrest via an ATM/p53-Related Pathway in Human Gastric Epithelium GES-1 Cells In Vitro

Sterigmatocystin (ST), which is commonly detected in food and feed commodities, is a mutagenic and carcinogenic mycotoxin that has been recognized as a possible human carcinogen. Our previous study showed that ST can induce G2 phase arrest in GES-1 cells in vitro and that the MAPK and PI3K signaling pathways are involved in the ST-induced G2 arrest. It is now widely accepted that DNA damage plays a critical role in the regulation of cell cycle arrest and apoptosis. In response to DNA damage, a complex signaling network is activated in eukaryotic cells to trigger cell cycle arrest and facilitate DNA repair. To further explore the molecular mechanism through which ST induces G2 arrest, the current study was designed to precisely dissect the role of DNA damage and the DNA damage sensor ataxia telangiectasia-mutated (ATM)/p53-dependent pathway in the ST-induced G2 arrest in GES-1 cells. Using the comet assay, we determined that ST induces DNA damage, as evidenced by the formation of DNA comet tails, in GES-1 cells. We also found that ST induces the activation of ATM and its downstream molecules, Chk2 and p53, in GES-1 cells. The ATM pharmacological inhibitor caffeine was found to effectively inhibit the activation of the ATM-dependent pathways and to rescue the ST-induced G2 arrest in GES-1 cells, which indicating its ATM-dependent characteristic. Moreover, the silencing of the p53 expression with siRNA effectively attenuated the ST-induced G2 arrest in GES-1 cells. We also found that ST induces apoptosis in GES-1 cells. Thus, our results show that the ST-induced DNA damage activates the ATM/53-dependent signaling pathway, which contributes to the induction of G2 arrest in GES-1 cells.

Identification of Shigella flexneri IcsA residues affecting interaction with N-WASP, and evidence for IcsA-IcsA co-operative interaction.

The Shigella flexneri IcsA (VirG) protein is a polarly distributed outer membrane protein that is a fundamental virulence factor which interacts with neural Wiskott-Aldrich syndrome protein (N-WASP). The activated N-WASP then activates the Arp2/3 complex which initiates de novo actin nucleation and polymerisation to form F-actin comet tails and allows bacterial cell-to-cell spreading. In a previous study, IcsA was found to have three N-WASP interacting regions (IRs): IR I (aa 185–312), IR II (aa 330–382) and IR III (aa 508–730). The aim of this study was to more clearly define N-WASP interacting regions II and III by site-directed mutagenesis of specific amino acids. Mutant IcsA proteins were expressed in both smooth lipopolysaccharide (S-LPS) and rough LPS (R-LPS) S. flexneri strains and characterised for IcsA production level, N-WASP recruitment and F-actin comet tail formation. We have successfully identified new amino acids involved in N-WASP recruitment within different N-WASP interacting regions, and report for the first time using co-expression of mutant IcsA proteins, that N-WASP activation involves interactions with different regions on different IcsA molecules as shown by Arp3 recruitment. In addition, our findings suggest that autochaperone (AC) mutant protein production was not rescued by another AC region provided in trans, differing to that reported for two other autotransporters, PrtS and BrkA autotransporters.

Clusters of Arp2/3 Activators Mimic Pathogenic Actin Comets and Pedestals

We investigate RTK signaling to the actin cytoskeleton through the Nck adaptor protein. The SH2 domain of Nck binds phosphorylated tyrosine residues and the SH3 domains of Nck bind and activate N-WASP. Activation of N-WASP involves release of the VCA domain. Simultaneous binding of G-actin monomers and actin nucleation factor Arp2/3 to the VCA domain initiates nucleation of a new actin branch. Pathogenic microorganisms induce this pathway to exploit actin polymerization machinery of the host.Experimental aggregation of a fusion protein containing the Nck SH3 domains on the plasma membrane results in the formation of dynamic actin comet tails. The VCA domain of N-WASP is directly responsible for Arp2/3 activation, and aggregation of VCA can bypass the need for adaptor proteins such as Nck to initiate actin polymerization. We used image analysis to characterize the differences between Nck- and VCA-induced actin structures. Morphometric analysis demonstrates that aggregation of VCA domains on the membrane produced thicker, denser and less elongated actin structures. Particle tracking showed that motile Nck comets move faster than VCA-induced actin structures. Our data indicates that Nck comets reproduce the behavior of Vaccinia comets and VCA clusters resemble EPEC/EHEC actin pedestals. As a consequence of the experimental design, VCA membrane clusters have higher VCA density than Nck clusters. We therefore co-aggregated trans-membrane VCA domains with “dummy” fusion proteins. However, morphology and dynamics of VCA actin structures did not alter significantly, suggesting density alterations cannot explain differences between Nck and VCA-induced structures. We are now testing whether higher VCA turnover and additional Nck binding proteins could explain the more dynamic nature of Nck-induced structures. Understanding the basis for the differences between the Nck and VCA induced actin assemblies will advance our knowledge of RTK signaling to mobilize actin cytoskeleton.

Absence of O antigen suppresses Shigella flexneri IcsA autochaperone region mutations

The Shigella flexneri IcsA (VirG) protein is a polarly distributed autotransporter protein. IcsA functions as a virulence factor by interacting with the host actin regulatory protein N-WASP, which in turn activates the Arp2/3 complex, initiating actin polymerization. Formation of F-actin comet tails allows bacterial cell-to-cell spreading. Although various accessory proteins such as periplasmic chaperones and the β-barrel assembly machine (BAM) complex have been shown to be involved in the export of IcsA, the IcsA translocation mechanism remains to be fully elucidated. A putative autochaperone (AC) region (amino acids 634-735) located at the C-terminal end of the IcsA passenger domain, which forms part of the self-associating autotransporter (SAAT) domain, has been suggested to be required for IcsA biogenesis, as well as for N-WASP recruitment, based on mutagenesis studies. IcsA(i) proteins with linker insertion mutations within the AC region have a significant reduction in production and are defective in N-WASP recruitment when expressed in smooth LPS (S-LPS) S. flexneri. In this study, we have found that the LPS O antigen plays a role in IcsA(i) production based on the use of an rmlD (rfbD) mutant having rough LPS (R-LPS) and a novel assay in which O antigen is depleted using tunicamycin treatment and then regenerated. In addition, we have identified a new N-WASP binding/interaction site within the IcsA AC region.

Influence of chloroquine on kinetics of single-cell gel electrophoresis

In single-cell gel electrophoresis (the comet assay) the DNA of lysed cells, the nucleoids, extends towards the anode in a track resembling a comet tail. The aim of this work was to investigate the effects of changes in DNA topology on this process. Methods. We used the kinetic approach, proposed earlier by us, to measure a relative amount of DNA in the comet tails as a function of time in the presence of different concentrations of chloroquine, a widely used intercalator. Results. We have shown that, at given small concentrations, intercalation of chloroquine strongly facilitates the comet tail formation. At the same time, some part of DNA (about 8 %) in the nucleoids exits very fast independently on chloroquine, while the largest part of DNA (about three quarters) does not exit at all. At high concentrations the intercalator increases the fraction of DNA, which cannot exit. Conclusions. Our results imply that the loop domains, which contain about one to several hundreds kilobases, represent only a small part (about a quarter) of DNA in the nucleus. The intercalation induces detachment of these loops from the nuclear matrix.

Comet‐FISH with rDNA probes for the analysis of mutagen‐induced DNA damage in plant cells

We used comet-fluorescence in situ hybridization (FISH) in the model plant species Crepis capillaris following exposure of seedlings to maleic hydrazide (MH). FISH with 5S and 25S rDNA probes was applied to comets obtained under alkaline conditions to establish whether these DNA regions were preferentially involved in comet tail formation. MH treatment induced significant fragmentation of nuclear DNA and of rDNA loci. A 24-h post-treatment recovery period allowed a partial reversibility of MH-induced damage on nuclear and rDNA regions. Analyses of FISH signals demonstrated that rDNA sequences were always involved in tail formation and that 5S rDNA was more frequently present in the tail than 25S rDNA, regardless of treatment. The involvement of 25S rDNA in nucleolus formation and differences in chromatin structure between the two loci may explain the different susceptibility of the 25S and 5S rDNA regions to migrate into the tail. This work is the first report on the application of FISH to comet preparations from plants to analyze the distribution and repair of DNA damage within specific genomic regions after mutagenic treatment. Moreover, our work suggests that comet-FISH in plants may be a useful tool for environmental monitoring assessment.

Nexilin is a dynamic component of Listeria monocytogenes and enteropathogenic Escherichia coli actin-rich structures

The bacterial pathogens Listeria monocytogenes and enteropathogenic Escherichia coli (EPEC) generate motile actin-rich structures (comet tails and pedestals) as part of their infectious processes. Nexilin, an actin-associated protein and a component of focal adhesions, has been suggested to be involved in actin-based motility. To determine whether nexilin is commandeered during L. monocytogenes and EPEC infections, we infected cultured cells and found that nexilin is crucial for L. monocytogenes invasion as levels of internalized bacteria were significantly decreased in nexilin-targeted siRNA-treated cells. In addition, nexilin is a component of the machinery that drives the formation of L. monocytogenes comet tails and EPEC pedestals. Nexilin colocalizes with stationary bacteria and accumulates at the distal portion of comet tails and pedestals of motile bacteria. We also show that nexilin is crucial for efficient comet tail formation as cells pre-treated with nexilin siRNA generate malformed comet tails, whereas nexilin is dispensable during EPEC pedestal generation. These findings demonstrate that nexilin is required for efficient infection with invasive and adherent bacteria and is key to the actin-rich structures these microbes generate.

How Is the Formation of Comet Tails Supported by the Incision Step of Nucleotide Excision Repair?

How Is the Formation of Comet Tails Supported by the Incision Step of Nucleotide Excision Repair?

Role of hesperetin (a natural flavonoid) and its analogue on apoptosis in HT-29 human colon adenocarcinoma cell line – A comparative study

Colon cancer is one of the serious health problems in most developed countries and its incidence rate is increasing in India. Hesperetin (HN) (3′,5,7-trihydroxy-4′-methoxyflavonone) and hesperetin analogue (HA) were tested for their apoptosis inducing ability. Methyl thiazolyl tetrazolium assay revealed a dose as well as duration-dependent reduction of HT-29 (colon adenocarcinoma) cellular growth in response to HN and HA treatment. At 24h 70μM of HN and 32μM of HA showed 50% reduction of HT-29 cellular growth. Acridine orange/ethidium bromide staining showed apoptotic features of cell death induced by HN and HA. Rhodamine 123 staining showed significant reduction in mitochondrial membrane potential induced by HN and HA. HN and HA induced DNA damage was confirmed by comet tail formation. Lipid peroxidation markers (TBARS) and protein oxidation marker (PCC) were significantly elevated in HN and HA treated groups. Enzymic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) were slightly decreased in their activities compared to control (untreated HT-29 cells). Results of Western blot analysis of apoptosis associated genes revealed an increase in cytochrome C, Bax, cleaved caspase-3 expression and a decrease in Bcl-2 expression. These findings indicate that HN and HA induce apoptosis on HT-29 via Bax dependent mitochondrial pathway involving oxidant/antioxidant imbalance.

The kinetochore protein Bub1 participates in the DNA damage response

The DNA damage response (DDR) and the spindle assembly checkpoint (SAC) are two critical mechanisms by which mammalian cells maintain genome stability. There is a growing body of evidence that DDR elements and SAC components crosstalk. Here we report that Bub1 ( budding uninhibited by benzimidazoles 1), one of the critical kinetochore proteins essential for SAC, is required for optimal DDRs. We found that knocking-down Bub1 resulted in prolonged H2AX foci and comet tail formation as well as hypersensitivity in response to ionizing radiation (IR). Further, we found that Bub1-mediated Histone H2A Threonine 121 phosphorylation was induced after IR in an ATM-dependent manner. We demonstrated that ATM phosphorylated Bub1 on serine 314 in response to DNA damage in vivo. Finally, we showed that ATM-mediated Bub1 serine 314 phosphorylation was required for IR-induced Bub1 activation and for the optimal DDR. Together, we elucidate the molecular mechanism of DNA damage-induced Bub1 activation and highlight a critical role of Bub1 in DDR.

The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis

Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium ΔsipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress.

Nck Function in Tyrosine Kinase Signaling to the Actin Cytoskeleton

Tyrosine kinase signaling leads to the post-translational modification of proteins and their binding partners. These modifications lead to the membrane recruitment of signaling proteins, promoting an increase in their local concentration, which results in a cellular response to the phosphorylation of tyrosine residues. Nck, an SH2/SH3 adaptor protein, functions in tyrosine kinase signaling by linking tyrosine phosphorylation on the membrane with binding partners, such as N-WASp, that function in facilitating actin nucleation and polymerization. Recent in vitro studies have demonstrated that N-WASp dimers bind to and activate N-WASp far more efficiently than individual N-WASp molecules. Because Nck has been shown to induce actin comet tail formation in an N-WASp dependent manner, we manipulate this molecular interaction to test the effect of N-WASp dimerization on actin polymerization. To manipulate the pathway, we vary the concentration of Nck within antibody-induced aggregates on the cellular surface and then use precise quantitative analysis of Nck density and induced actin polymerization to analyze the system. Using the Virtual Cell, we have built a comprehensive, quantitative actin cycle model. With this model we have tested 2 mathematical expressions for dimerized N-WASp activation of Arp2/3, which produce different experimentally testable predictions. These results demonstrate a nonlinear relationship between the density of Nck aggregates and actin polymerization and also demonstrate that a quadratic-based formula describing dimerized N-WASp activation of Arp2/3 most accurately predicts experimental observations. The combination of biochemical modeling and precise experimental manipulation and quantification provides unique insights into the relationship between the density of increased local concentrations of Nck and resulting localized actin polymerization. (Supported by NIH grants P41 RR013186 and U54 RR022232)

EZH2 Promotes Expansion of Breast Tumor Initiating Cells through Activation of RAF1-β-Catenin Signaling

It has been proposed that an aggressive secondary cancer stem cell population arises from a primary cancer stem cell population through acquisition of additional genetic mutations and drives cancer progression. Overexpression of Polycomb protein EZH2, essential in stem cell self-renewal, has been linked to breast cancer progression. However, critical mechanism linking increased EZH2 expression to BTIC (breast tumor initiating cell) regulation and cancer progression remains unclear. Here, we identify a mechanism in which EZH2 expression-mediated downregulation of DNA damage repair leads to accumulation of recurrent RAF1 gene amplification in BTICs, which activates p-ERK-β-catenin signaling to promote BTIC expansion. We further reveal that AZD6244, a clinical trial drug that inhibits RAF1-ERK signaling, could prevent breast cancer progression by eliminating BTICs.

Formin' a comet tail

Comet tail formation in Rickettsia spp. relies on the formin-like protein Sca2 to mediate actin nucleation.

Relative actin nucleation promotion efficiency by WASP and WAVE proteins in endothelial cells

The mammalian genome encodes multiple Wiskott–Aldrich syndrome protein (WASP)/WASP-family Verprolin homologous (WAVE) proteins. Members of this family interact with the actin related protein (Arp) 2/3 complex to promote growth of a branched actin network near the plasma membrane or the surface of moving cargos. Arp2/3 mediated branching can further lead to formation of comet tails (actin rockets). Despite their similar domain structure, different WASP/WAVE family members fulfill unique functions that depend on their subcellular location and activity levels. We measured the relative efficiency of actin nucleation promotion of full-length WASP/WAVE proteins in a cytoplasmic extract from primary human umbilical vein endothelial cells (HUVEC). In this assay WAVE2 and WAVE3 complexes showed higher nucleation efficiency than WAVE1 and N-WASP, indicating distinct cellular controls for different family members. Previously, WASP and N-WASP were the only members that were known to stimulate comet formation. We observed that in addition to N-WASP, WAVE3 also induced short actin tails, and the other WAVEs induced formation of asymmetric actin shells. Differences in shape and structure of actin-based growth may reflect varying ability of WASP/WAVE proteins to break symmetry of the actin shell, possibly by differential recruitment of actin bundling or severing (pruning or debranching) factors.

347 The inflammatory tumour microenvironment: tumour-protective or tumour promoting?

The DNA damage response (DDR) and the spindle assembly checkpoint (SAC) are two critical mechanisms by which mammalian cells maintain genome stability. There is a growing body of evidence that DDR elements and SAC components crosstalk. Here we report that Bub1 (budding uninhibited by benzimidazoles 1), one of the critical kinetochore proteins essential for SAC, is required for optimal DDRs. We found that knocking-down Bub1 resulted in prolonged H2AX foci and comet tail formation as well as hypersensitivity in response to ionizing radiation (IR). Further, we found that Bub1-mediated Histone H2A Threonine 121 phosphorylation was induced after IR in an ATM-dependent manner. We demonstrated that ATM phosphorylated Bub1 on serine 314 in response to DNA damage in vivo. Finally, we showed that ATM-mediated Bub1 serine 314 phosphorylation was required for IR-induced Bub1 activation and for the optimal DDR. Together, we elucidate the molecular mechanism of DNA damage-induced Bub1 activation and highlight a critical role of Bub1 in DDR.

Fisetin induces apoptosis in Huh-7 cells via downregulation of BIRC8 and Bcl2L2

This study aimed to investigate the effects of fisetin, a common dietary natural flavonoid, on apoptosis of Huh-7 cells. The MTT assay was used to evaluate the reduction of cell viability. In the DNA fragmentation assay and comet assay, fisetin-induced DNA damage was visible as a formation of DNA fragmentation and comet tails. Fisetin also induced intracellular accumulation of reactive oxygen species. Two-dimensional gel electrophoresis was performed to evaluate protein expression in fisetin-treated and control cells, and Baculoviral IAP repeat-containing protein 8 (BIRC8) and apoptosis regulator Bcl-W (Bcl2L2) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their expression was confirmed by western blotting. The anticancer effect of fisetin in Huh-7 cells may result from the down regulation of BIRC8 and the Bcl2L2.