The α-tubulin acetyltransferase ATAT1: structure, cellular functions, and its emerging role in human diseases.

The acetylation of α-tubulin on lysine 40 is a well-studied post-translational modification which has been associated with the presence of long-lived stable microtubules that are more resistant to mechanical breakdown. The discovery of α-tubulin acetyltransferase 1 (ATAT1), the enzyme responsible for lysine 40 acetylation on α-tubulin in a wide range of species, including protists, nematodes, and mammals, dates to about a decade ago. However, the role of ATAT1 in different cellular activities and molecular pathways has been only recently disclosed. This review comprehensively summarizes the most recent knowledge on ATAT1 structure and substrate binding and analyses the involvement of ATAT1 in a variety of cellular processes such as cell motility, mitosis, cytoskeletal organization, and intracellular trafficking. Finally, the review highlights ATAT1 emerging roles in human diseases and discusses ATAT1 potential enzymatic and non-enzymatic roles and the current efforts in developing ATAT1 inhibitors.

ALFI: Cell cycle phenotype annotations of label-free time-lapse imaging data from cultured human cells.

Detecting and tracking multiple moving objects in a video is a challenging task. For living cells, the task becomes even more arduous as cells change their morphology over time, can partially overlap, and mitosis leads to new cells. Differently from fluorescence microscopy, label-free techniques can be easily applied to almost all cell lines, reducing sample preparation complexity and phototoxicity. In this study, we present ALFI, a dataset of images and annotations for label-free microscopy, made publicly available to the scientific community, that notably extends the current panorama of expertly labeled data for detection and tracking of cultured living nontransformed and cancer human cells. It consists of 29 time-lapse image sequences from HeLa, U2OS, and hTERT RPE-1 cells under different experimental conditions, acquired by differential interference contrast microscopy, for a total of 237.9 hours. It contains various annotations (pixel-wise segmentation masks, object-wise bounding boxes, tracking information). The dataset is useful for testing and comparing methods for identifying interphase and mitotic events and reconstructing their lineage, and for discriminating different cellular phenotypes.

Blockage of autophagosome-lysosome fusion through SNAP29 O-GlcNAcylation promotes apoptosis via ROS production.

Macroautophagy/autophagy has been shown to exert a dual role in cancer i.e., promoting cell survival or cell death depending on the cellular context and the cancer stage. Therefore, development of potent autophagy modulators, with a clear mechanistic understanding of their target action, has paramount importance in both mechanistic and clinical studies. In the process of exploring the mechanism of action of a previously identified cytotoxic small molecule (SM15) designed to target microtubules and the interaction domain of microtubules and the kinetochore component NDC80/HEC1, we discovered that the molecule acts as a potent autophagy inhibitor. By using several biochemical and cell biology assays we demonstrated that SM15 blocks basal autophagic flux by inhibiting the fusion of correctly formed autophagosomes with lysosomes. SM15-induced autophagic flux blockage promoted apoptosis-mediated cell death associated with ROS production. Interestingly, autophagic flux blockage, apoptosis induction and ROS production were rescued by genetic or pharmacological inhibition of OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) or by expressing an O-GlcNAcylation-defective mutant of the SNARE fusion complex component SNAP29, pointing to SNAP29 as the molecular target of SM15 in autophagy. Accordingly, SM15 was found to enhance SNAP29 O-GlcNAcylation and, thereby, inhibit the formation of the SNARE fusion complex. In conclusion, these findings identify a new pathway in autophagy connecting O-GlcNAcylated SNAP29 to autophagic flux blockage and autophagosome accumulation, that, in turn, drives ROS production and apoptotic cell death. Consequently, modulation of SNAP29 activity may represent a new opportunity for therapeutic intervention in cancer and other autophagy-associated diseases.

Pharmacological targeting of CBP/p300 drives a redox/autophagy axis leading to senescence-induced growth arrest in non-small cell lung cancer cells.

p300/CBP histone acetyltransferases (HAT) are critical transcription coactivators involved in multiple cellular activities. They act at multiple levels in non-small cell lung carcinoma (NSCLC) and appear, therefore, as promising druggable targets. Herein, we investigated the biological effects of A-485, the first selective (potent) drug-like HAT catalytic inhibitor of p300/CBP, in human NSCLC cell lines. A-485 treatment specifically reduced p300/CBP-mediated histone acetylation marks and caused growth arrest of lung cancer cells via activation of the autophagic pathway. Indeed, A-485 growth-arrested cells displayed phenotypic markers of cell senescence and failed to form colonies. Notably, disruption of autophagy by genetic and pharmacological approaches triggered apoptotic cell death. Mechanistically, A-485-induced senescence occurred through the accumulation of reactive oxygen species (ROS), which in turn resulted in DNA damage and activation of the autophagic pathway. Interestingly, ROS scavengers were able to revert senescence phenotype and restore cell viability, suggesting that ROS production had a key role in upstream events leading to growth arrest commitment. Altogether, our data provide new insights into the biological effects of the A-485 and uncover the importance of the autophagic/apoptotic response to design a new combinatorial anticancer strategy.

M2 Muscarinic Receptor Activation Impairs Mitotic Progression and Bipolar Mitotic Spindle Formation in Human Glioblastoma Cell Lines.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. METHODS: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression. RESULTS: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. CONCLUSIONS: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.

The Tubulin Code and Tubulin-Modifying Enzymes in Autophagy and Cancer.

Microtubules are key components of the cytoskeleton of eukaryotic cells. Microtubule dynamic instability together with the "tubulin code" generated by the choice of different α- and ß- tubulin isoforms and tubulin post-translational modifications have essential roles in the control of a variety of cellular processes, such as cell shape, cell motility, and intracellular trafficking, that are deregulated in cancer. In this review, we will discuss available evidence that highlights the crucial role of the tubulin code in determining different cancer phenotypes, including metastatic cell migration, drug resistance, and tumor vascularization, and the influence of modulating tubulin-modifying enzymes on cancer cell survival and aggressiveness. We will also discuss the role of post-translationally modified microtubules in autophagy-the lysosomal-mediated cellular degradation pathway-that exerts a dual role in many cancer types, either promoting or suppressing cancer growth. We will give particular emphasis to the role of tubulin post-translational modifications and their regulating enzymes in controlling the different stages of the autophagic process in cancer cells, and consider how the experimental modulation of tubulin-modifying enzymes influences the autophagic process in cancer cells and impacts on cancer cell survival and thereby represents a new and fruitful avenue in cancer therapy.

The Mitotic Apparatus and Kinetochores in Microcephaly and Neurodevelopmental Diseases.

Regulators of mitotic division, when dysfunctional or expressed in a deregulated manner (over- or underexpressed) in somatic cells, cause chromosome instability, which is a predisposing condition to cancer that is associated with unrestricted proliferation. Genes encoding mitotic regulators are growingly implicated in neurodevelopmental diseases. Here, we briefly summarize existing knowledge on how microcephaly-related mitotic genes operate in the control of chromosome segregation during mitosis in somatic cells, with a special focus on the role of kinetochore factors. Then, we review evidence implicating mitotic apparatus- and kinetochore-resident factors in the origin of congenital microcephaly. We discuss data emerging from these works, which suggest a critical role of correct mitotic division in controlling neuronal cell proliferation and shaping the architecture of the central nervous system.

A novel resveratrol derivative induces mitotic arrest, centrosome fragmentation and cancer cell death by inhibiting γ-tubulin.

BACKGROUND: Resveratrol and its natural stilbene-containing derivatives have been extensively investigated as potential chemotherapeutic agents. The synthetic manipulation of the stilbene scaffold has led to the generation of new analogues with improved anticancer activity and better bioavailability. In the present study we investigated the anticancer activity of a novel trimethoxystilbene derivative (3,4,4'-trimethoxylstilbene), where two methoxyl groups are adjacent on the benzene ring (ortho configuration), and compared its activity to 3,5,4'-trimethoxylstilbene, whose methoxyl groups are in meta configuration. RESULTS: We provide evidence that the presence of the two methoxyl groups in ortho configuration renders 3,4,4'-trimethoxystilbene more efficient than the meta isomer in inhibiting cell proliferation and producing apoptotic death in colorectal cancer cells. Confocal microscopy of α- and γ-tubulin staining shows that the novel compound strongly depolymerizes the mitotic spindle and produces fragmentation of the pericentrosomal material. Computer assisted docking studies indicate that both molecules potentially interact with γ-tubulin, and that 3,4,4'-trimethoxystilbene is likely to establish stronger interactions with the protein. CONCLUSIONS: These findings demonstrate the ortho configuration confers higher specificity for γ-tubulin with respect to α-tubulin on 3,4,4' trimethoxystilbene, allowing it to be defined as a new γ-tubulin inhibitor. A strong interaction with γ-tubulin might be a defining feature of molecules with high anticancer activity, as shown for the 3,4,4' isomer.

Molecular cytogenetics of the micronucleus: Still surprising.

The purpose of this review is to provide an historical overview of the molecular cytogenetic approaches that have been pursed to identify the mechanism of origin of micronuclei as well as highlight the significance of the findings obtained for the understanding of the molecular action of harmful agents in the micronucleus assay. Then, we summarize recent exciting findings that have been obtained by applying molecular approaches to analyze the biology of micronuclei. These findings emphasize the role of micronuclei in the generation of genomic instability in the form of complex localized rearrangements characteristic of chromothripsis/chromoanagenesis, and put in evidence a fundamental and yet poorly understood aspect of the cell response to micronuclei: the activation of a cell-intrinsic innate immune response.

The resveratrol analogue trimethoxystilbene inhibits cancer cell growth by inducing multipolar cell mitosis.

Natural compounds are extensively studied for their potential use in traditional and non-traditional medicine. Several natural and synthetic Resveratrol analogues have shown interesting biological activities in the field of cancer chemoprevention. In the present study, we have focused on the ability of Resveratrol and two methoxylated derivatives (Trimethoxystilbene and Pterostilbene) to inhibit human cancer cell growth particularly analyzing their ability to interfere with tubulin dynamics at mitosis. We show that Trimethoxystilbene, differently from Resveratrol and Pterostilbene, alters microtubule polymerization dynamics in HeLa cells specifically inducing multipolar spindles and mitotic arrest coupled to a reduction of cell growth and an increase in apoptotic death by mitotic catastrophe. This work demonstrates that the structural modification of Rsv causes substantial changes in the mechanism of action of the derivatives. The presence of three extra methyl groups renders Trimethoxy very efficient in impairing cell proliferation by inducing mitotic catastrophe in cancer cells. © 2016 Wiley Periodicals, Inc.

Xanthium strumarium extract inhibits mammalian cell proliferation through mitotic spindle disruption mediated by xanthatin.

ETHNOPHARMACOLOGICAL RELEVANCE: Xanthium strumarium L. is a member of the Asteraceae family popularly used with multiple therapeutic purposes. Whole extracts of this plant have shown anti-mitotic activity in vitro suggesting that some components could induce mitotic arrest in proliferating cells. AIM OF THE SUDY: Aim of the present work was to characterize the anti-mitotic properties of the X. strumarium whole extract and to isolate and purify active molecule(s). MATERIALS AND METHODS: The capacity of the whole extract to inhibit mitotic progression in mammalian cultured cells was investigated to identify its anti-mitotic activity. Isolation of active component(s) was performed using a bioassay-guided multistep separation procedure in which whole extract was submitted to a progressive process of fractionation and fractions were challenged for their anti-mitotic activity. RESULTS: Our results show for the first time that X. strumarium whole extract inhibits assembly of the mitotic spindle and spindle-pole separation, thereby heavily affecting mitosis, impairing the metaphase to anaphase transition and inducing apoptosis. The purification procedure led to a fraction with an anti-mitotic activity comparable to that of the whole extract. Chemical analysis of this fraction showed that its major component was xanthatin. CONCLUSIONS: The present work shows a new activity of X. strumarium extract, i.e. the alteration of the mitotic apparatus in cultured cells that may be responsible for the anti-proliferative activity of the extract. Anti-mitotic activity is shown to be mainly exerted by xanthatin.

Resveratrol affects DNA damage induced by ionizing radiation in human lymphocytes in vitro.

Resveratrol (3,4',5-trihydroxystilbene; RSV) acts on cancer cells in several ways, inducing cell cycle delay and apoptotic death, and enhancing ionizing radiation (IR)-mediated responses. However, fewer studies have examined RSV effects on normal cells. We have treated human lymphocytes in vitro with RSV, either alone or combined with IR, to evaluate its potential use as a radioprotector. We measured the effects of RSV on induction of DNA damage, repair kinetics, and modulation of histone deacetylase activity.

Erratum: Kinetochore-microtube attachments in cancer therapy.

[This corrects the article on p. 902 in vol. 2, PMID: 26697517.].

Resveratrol and its methoxy-derivatives as modulators of DNA damage induced by ionising radiation.

Various naturally occurring stilbene-like compounds that are related to resveratrol (RSV) possess some of the beneficial effects of the parent molecule and provide even further benefits. Therefore, a series of methoxylated analogues of RSV were prepared with the aim of increasing antitumour and proapoptotic activity. In a previous article, we studied two methoxy-derivatives, pterostilbene (PTERO) and trimethoxystilbene (TRIMETHOXY), in which the first was formed by the substitution of two hydroxyl groups with two methoxy groups (trans-3,5-dimethoxy-4'-hydroxystilbene) and the second was formed by the replacement of all three OH groups with methoxy groups (trans-3,5,4'-trimethoxystilbene). Both methoxy-derivatives showed stronger antioxidant activity when compared with RSV. In the present article, we focused on the analysis of the ability of RSV and its two methoxylated derivatives to protect proliferating non-tumoural cells from the damage induced by ionising radiation (IR). First we showed that the methoxy derivatives, contrary to their parental compound, are unable to affect topoisomerase enzyme and consequently are not clastogenic per se Second we showed that both PTERO and TRIMETHOXY more efficiently reduce the chromosome damage induced by IR. Furthermore, TRIMETHOXY, but not PTERO, causes a delay in cell proliferation, particularly in mitosis progression increasing the number of cells in metaphase at the expense of prophases and ana/telophases.

Tetraploid cells produced by absence of substrate adhesion during cytokinesis are limited in their proliferation and enter senescence after DNA replication.

Tetraploidy has been proposed as an intermediate state in neoplastic transformation due to the intrinsic chromosome instability of tetraploid cells. Despite the identification of p53 as a major factor in growth arrest of tetraploid cells, it is still unclear whether the p53-dependent mechanism for proliferation restriction is intrinsic to the tetraploid status or dependent on the origin of tetraploidy. Substrate adherence is fundamental for cytokinesis completion in adherent untransformed cells. Here we show that untransformed fibroblast cells undergoing mitosis in suspension produce binucleated tetraploid cells due to defective cleavage furrow constriction that leads to incomplete cell abscission. Binucleated cells obtained after loss of substrate adhesion maintain an inactive p53 status and are able to progress into G1 and S phase. However, binucleated cells arrest in G2, accumulate p53 and are not able to enter mitosis as no tetraploid metaphases were recorded after one cell cycle time. In contrast, tetraploid metaphases were found following pharmacological inhibition of Chk1 kinase, suggesting the involvement of the ATR/Chk1 pathway in the G2 arrest of binucleated cells. Interestingly, after persistence in the G2 phase of the cell cycle, a large fraction of binucleated cells become senescent. These findings identify a new pathway of proliferation restriction for tetraploid untransformed cells that seems to be specific for loss of adhesion-dependent cytokinesis failure. This involves Chk1 and p53 activation during G2. Inhibition of growth and entrance into senescence after cytokinesis in suspension may represent an important mechanism to control tumor growth. In fact, anchorage independent growth is a hallmark of cancer and it has been demonstrated that binucleated transformed cells can enter a cycle of anchorage independent growth.

Cytotoxic and genotoxic effects mediated by M2 muscarinic receptor activation in human glioblastoma cells.

Glioblastomas are the most common brain tumors in humans. Previously, we demonstrated that the muscarinic receptor agonist, arecaidine propargyl ester, via M2 receptors, inhibits cell proliferation in a time and dose-dependent manner and induces a severe apoptosis in human U251 and U87 glioblastoma cell lines. In order to clarify the mechanisms causing apoptosis after arecaidine treatment, we analyzed the ability of arecaidine to induce oxidative stress. By dichloro-dihydro-fluorescein diacetate (DCFDA) staining, we demonstrated that arecaidine increased the intracellular ROS levels. ROS accumulation was completely counteracted by the ROS scavenger, N-acetyl-l-cysteine (NAC). Apoptotic cell death appeared directly correlated to ROS production since NAC was able to counteract this effect. Although there was an up-regulation of some detoxifying enzyme expression such as superoxide dismutase (MnSOD) and sirtuin-1 (SIRT1), the cytotoxic effect caused by arecaidine treatment caused DNA damage, as demonstrated by the increase of histone γ-H2AX positive cells, and chromosomal aberrations. These effects were mediated by M2 receptor activation; in fact after silencing of M2 receptors by siRNA, the increase of γ-H2AX positive cells was abolished. In conclusion, in addition to a cytostatic effect previously described, in the present study we have better characterized the mechanisms causing the cytotoxic effects and the apoptotic cell death in glioblastoma cells after M2 receptor activation. These data allow to consider this receptor a new interesting therapeutic tool for the glioblastoma treatment.

Genomic instability: Crossing pathways at the origin of structural and numerical chromosome changes.

Genomic instability leads to a wide spectrum of genetic changes, including single nucleotide mutations, structural chromosome alterations, and numerical chromosome changes. The accepted view on how these events are generated predicts that separate cellular mechanisms and genetic events explain the occurrence of these types of genetic variation. Recently, new findings have shed light on the complexity of the mechanisms leading to structural and numerical chromosome aberrations, their intertwining pathways, and their dynamic evolution, in somatic as well as in germ cells. In this review, we present a critical analysis of these recent discoveries in this area, with the aim to contribute to a deeper knowledge of the molecular networks leading to adverse outcomes in humans following exposure to environmental factors. The review illustrates how several technological advances, including DNA sequencing methods, bioinformatics, and live-cell imaging approaches, have contributed to produce a renewed concept of the mechanisms causing genomic instability. Special attention is also given to the specific pathways causing genomic instability in mammalian germ cells. Remarkably, the same scenario emerged from some pioneering studies published in the 1980s to 1990s, when the evolution of polyploidy, the chromosomal effects of spindle poisons, the fate of micronuclei, were intuitively proposed to share mechanisms and pathways. Thus, an old working hypothesis has eventually found proper validation.

Xanthium strumarium L. extracts produce DNA damage mediated by cytotoxicity in in vitro assays but does not induce micronucleus in mice.

Xanthium strumarium L. is a member of the Asteraceae commonly used in Cuba, mainly as diuretic. Some toxic properties of this plant have also been reported and, to date, very little is known about its genotoxic properties. The present work aims was to evaluate the potential cytotoxic and genotoxic risk of whole extract from Xanthium strumarium L. whole extract of aerial parts. No positive response was observed in a battery of four Salmonella typhimurium strains, when exposed to concentrations up to 5 mg/plate, with and without mammalian metabolic activation (liver microsomal S9 fraction from Wistar rats). In CHO cells, high concentrations (25-100 µg/mL) revealed significant reduction in cell viability. Results from sister chromatid exchanges, chromosome aberrations, and comet assay showed that X. strumarium extract is genotoxic at the highest concentration used, when clear cytotoxic effects were also observed. On the contrary, no increase in micronuclei frequency in bone marrow cells was observed when the extract was orally administered to mice (100, 500, and 2000 mg/Kg doses). The data presented here constitute the most complete study on the genotoxic potential of X. strumarium L. and show that the extract can induce in vitro DNA damage at cytotoxic concentrations.

Benzo[a]pyrene-induced cell cycle arrest in HepG2 cells is associated with delayed induction of mitotic instability.

The environmental carcinogen benzo[a]pyrene (B[a]P) after being metabolised by cytochrome P450 enzymes forms DNA adducts. This abnormal situation induces changes in the cell cycle, DNA damage, chromosomal and mitotic aberrations, all of which may be related to carcinogenesis. In order to further investigate the mechanistic basis of these effects, HepG2 cells were treated with 3µM B[a]P for various time periods, followed by further incubation in the absence of B[a]P for up to 192h. B[a]P treatment led initially to S-phase arrest followed by recovery and subsequent induction of G2/M arrest, indicating activation of the corresponding DNA damage checkpoints. Immunofluorescence-based studies revealed accumulation of B[a]P-induced DNA adducts and chromosomal damage which persisted beyond mitosis and entry into a new cycle, thus giving rise to a new round of activation of the S-phase checkpoint. Prolonged further cultivation of the cells in the absence of B[a]P resulted in high frequencies of various abnormal mitotic events. Abrogation of the B[a]P-induced S-phase arrest by the Chk1 inhibitor UCN-01 triggered a strong apoptotic response but also dramatically decreased the frequency of mitotic abnormalities in the surviving cells, suggesting that events occurring during S-phase arrest contribute to the formation of delayed mitotic damage. Overall, our data demonstrate that, although S-phase arrest serves as a mechanism by which the cells reduce their load of genetic damage, its prolonged activation may also have a negative impact on the balance between cell death and heritable genetic damage.