Empirical single-cell tracking and cell-fate simulation reveal dual roles of p53 in tumor suppression.

The tumor suppressor p53 regulates various stress responses via increasing its cellular levels. The lowest p53 levels occur in unstressed cells; however, the functions of these low levels remain unclear. To investigate the functions, we used empirical single-cell tracking of p53-expressing (Control) cells and cells in which p53 expression was silenced by RNA interference (p53 RNAi). Here, we show that p53 RNAi cells underwent more frequent cell death and cell fusion, which further induced multipolar cell division to generate aneuploid progeny. Those results suggest that the low levels of p53 in unstressed cells indeed have a role in suppressing the induction of cell death and the formation of aneuploid cells. We further investigated the impact of p53 silencing by developing an algorithm to simulate the fates of individual cells. Simulation of the fate of aneuploid cells revealed that these cells could propagate to create an aneuploid cell population. In addition, the simulation also revealed that more frequent induction of cell death in p53 RNAi cells under unstressed conditions conferred a disadvantage in terms of population expansion compared with Control cells, resulting in faster expansion of Control cells compared with p53 RNAi cells, leading to Control cells predominating in mixed cell populations. In contrast, the expansion of Control cells, but not p53 RNAi cells, was suppressed when the damage response was induced, allowing p53 RNAi cells to expand their population compared with the Control cells. These results suggest that, although p53 could suppress the formation of aneuploid cells, which could have a role in tumorigenesis, it could also allow the expansion of cells lacking p53 expression when the damage response is induced. p53 may thus play a role in both the suppression and the promotion of malignant cell formation during tumorigenesis.

Galectin-3 enhances neutrophil motility and extravasation into the airways during Aspergillus fumigatus infection.

Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian ß-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections.

Dose-dependent spatiotemporal responses of mammalian cells to an alkylating agent.

Cultured cell populations are composed of heterogeneous cells, and previous single-cell lineage tracking analysis of individual HeLa cells provided empirical evidence for significant heterogeneity of the rate of cell proliferation and induction of cell death. Nevertheless, such cell lines have been used for investigations of cellular responses to various substances, resulting in incomplete characterizations. This problem caused by heterogeneity within cell lines could be overcome by investigating the spatiotemporal responses of individual cells to a substance. However, no approach to investigate the responses by analyzing spatiotemporal data is currently available. Thus, this study aimed to analyze the spatiotemporal responses of individual HeLa cells to cytotoxic, sub-cytotoxic, and non-cytotoxic doses of the well-characterized carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although cytotoxic doses of MNNG are known to induce cell death, the single-cell tracking approach revealed that cell death occurred following at least four different cellular events, suggesting that cell death is induced via multiple processes. We also found that HeLa cells exposed to a sub-cytotoxic dose of MNNG were in a state of equilibrium between cell proliferation and cell death, with cell death again induced through different processes. However, exposure of cells to a non-cytotoxic dose of MNNG promoted growth by reducing the cell doubling time, thus promoting the growth of a sub-population of cells. A single-cell lineage tracking approach could dissect processes leading to cell death in a spatiotemporal manner and the results suggest that spatiotemporal data obtained by tracking individual cells can be used as a new type of bioinformatics data resource that enables the examination of cellular responses to various external substances.

Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), in which mutations in the dystrophin gene disrupts the firm adhesion. In patients with DMD, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in patients with DMD, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina and increases the efficiency of the myogenesis. Galectin-3 is an oligosaccharide-binding protein and is known to be involved in cell-cell interactions and cell-matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle, although its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the synthesis of binding partners (oligosaccharides) of galectin-3, promote myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased muscle-force production. The results suggest that treatment with N-acetylglucosamine might mitigate the burden of DMD.-Rancourt, A., Dufresne, S. S., St-Pierre, G., Lévesque, J.-C., Nakamura, H., Kikuchi, Y., Satoh, M. S., Frenette, J., Sato, S. Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny.

Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells.

Double-stranded DNA binding domain of poly(ADP-ribose) polymerase-1 and molecular insight into the regulation of its activity.

Poly(ADP-ribose) polymerase-1 (PARP-1) modifies various proteins, including itself, with ADP-ribose polymers (automodification). Polymer synthesis is triggered by binding of its zinc finger 1 (Zn1) and 2 (Zn2) to DNA breaks and is followed by inactivation through automodification. The multiple functional domains of PARP-1 appear to regulate activation and automodification-mediated inactivation of PARP-1. However, the roles of these domains in activation-inactivation processes are not well understood. Our results suggest that Zn1, Zn2, and a domain identified in this study, the double-stranded DNA binding (DsDB) domain, are involved in DNA break-dependent activation of PARP-1. We found that binding of the DsDB domain to double-stranded DNA and DNA break recognition by Zn1 and Zn2, whose actual binding targets are likely to be single-stranded DNA, lead to the activation of PARP-1. In turn, the displacement of single- and double-stranded DNA from Zn2 and the DsDB domain caused by ADP-ribose polymer synthesis results in the dissociation of PARP-1 from DNA breaks and thus its inactivation. We also found that the WGR domain is one of the domains involved in the RNA-dependent activation of PARP-1. Furthermore, because zinc finger 3 (Zn3) has the ability to bind to single-stranded RNA, it may have an indirect role in RNA-dependent activation. PARP-1 functional domains, which are involved in oligonucleic acid binding, therefore coordinately regulate PARP-1 activity depending on the status of the neighboring oligonucleic acids. Based on these results, we proposed a model for the regulation of PARP-1 activity.

Delocalization of nucleolar poly(ADP-ribose) polymerase-1 to the nucleoplasm and its novel link to cellular sensitivity to DNA damage.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme activated by binding to DNA breaks, which causes PARP-1 automodification. PARP-1 activation is required for regulating various cellular processes, including DNA repair and cell death induction. PARP-1 involved in these regulations is localized in the nucleoplasm, but approximately 40% of PARP-1 can be found in the nucleolus. Previously, we have reported that nucleolar PARP-1 is delocalized to the nucleoplasm in cells exposed to DNA-damaging agents. However, the functional roles of this delocalization in cellular response to DNA damage is not well understood, since this approach simultaneously induces the delocalization of PARP-1 and its automodification. We therefore devised an approach for separating these processes. Unmodified PARP-1 was first delocalized from the nucleolus using camptothecin. Then, PARP-1 was activated by exposure of cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In contrast to treatment with MNNG alone, delocalization of PARP-1 by CPT, prior to its activation by MNNG, induced extensive automodification of PARP-1. DNA repair activity and consumption of intracellular NAD(+) were not affected by this activation. On the other hand, activation led to an increased formation of apoptotic cells, and this effect was suppressed by inhibition of PARP-1 activity. These results suggest that delocalization of PARP-1 from the nucleolus to the nucleoplasm sensitizes cells to DNA damage-induced apoptosis. As it has been suggested that the nucleolus has a role in stress sensing, nucleolar PARP-1 could participate in a process involved in nucleolus-mediated stress sensing.

Induction of base damages representing a high risk site for double-strand DNA break formation in genomic DNA by exposure of cells to DNA damaging agents.

DNA repair is known as a defense mechanism against genotoxic insults. However, the most lethal type of DNA damages, double-strand DNA breaks (DSBs), can be produced by DNA repair. We have previously demonstrated that when long patch base excision repair attempts to repair a synthetic substrate containing two uracils, the repair produces DSBs (Vispe, S. and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392 and Vispe, S., Ho, E. L., Yung, T. M., and Satoh, M. S. (2003) J. Biol. Chem. 278, 35279-35285). In this synthetic substrate, the two uracils are located on the opposite DNA strands (separated by an intervening sequence stable at 37 degrees C) and represent a high risk site for DSB formation. It is not clear, however, whether similar high risk sites are also induced in genomic DNA by exposure to DNA damaging agents. Thus, to investigate the mechanisms of DSB formation, we have modified the DSB formation assay developed previously and demonstrated that high risk sites for DSB formation are indeed generated in genomic DNA by exposure of cells to alkylating agents. In fact, genomic DNA containing alkylated base damages, which could represent high risk sites, are converted into DSBs by enzymes present in extracts prepared from cells derived from clinically normal individuals. Furthermore, DSBs are also produced by extracts from cells derived from ataxia-telangiectasia patients who show cancer proneness due to an impaired response to DSBs. These results suggest the presence of a novel link between base damage formation and DSBs and between long patch base excision repair and human diseases that occur due to an impaired response to DSB.

Poly(ADP-ribose) polymerase-1 is a negative regulator of HIV-1 transcription through competitive binding to TAR RNA with Tat.positive transcription elongation factor b (p-TEFb) complex.

Human immunodeficiency virus, type 1 (HIV-1) transcription is regulated by a virus-encoded protein, Tat, which forms a complex with a host cellular factor, positive transcription elongation factor b (P-TEFb). When this complex binds to TAR RNA synthesized from the HIV-1 long terminal repeat promoter element, transcription is trans-activated. In this study we showed that, in host cells, HIV-1 transcription is negatively regulated by competition of poly(ADP-ribose) polymerase-1 (PARP-1) with Tat.P-TEFb for binding to TAR RNA. PARP-1, which has a high affinity for TAR RNA (K(D) = 1.35 x 10(-10) M), binds to the loop region of TAR RNA and displaces Tat or Tat.P-TEFb from the RNA. In vitro transcription assays showed that this displacement leads to suppression of Tat-mediated trans-activation of transcription. Furthermore in vivo expression of luciferase or destabilized enhanced green fluorescent protein genes under the control of the HIV-1 long terminal repeat promoter was suppressed by PARP-1. Thus, these results suggest that PARP-1 acts as a negative regulator of HIV-1 transcription through competitive binding with Tat or the Tat.P-TEFb complex to TAR RNA.

Poly(ADP-ribosyl)ation as a DNA damage-induced post-translational modification regulating poly(ADP-ribose) polymerase-1-topoisomerase I interaction.

Poly(ADP-ribosyl)ation is a post-translational modification that occurs immediately after exposure of cells to DNA damaging agents. In vivo, 90% of ADP-ribose polymers are attached to the automodification domain of poly(ADP-ribose) polymerase-1 (PARP-1), the main enzyme catalyzing this modification reaction. This enzyme forms complexes with transcription initiation, DNA replication, and DNA repair factors. In most known cases, the interactions occur through the automodification domain. However, functional implications of the automodification reaction on these interactions have not yet been elucidated. In the present study, we created fluorescent protein-tagged PARP-1 to study this enzyme in live cells and focused on the interaction between PARP-1 and topoisomerase I (Topo I), one of the enzymes that interacts with PARP-1 in vitro. Here, we demonstrate that PARP-1 co-localizes with Topo I throughout the cell cycle. Results from bioluminescence resonance energy transfer assays suggest that the co-localization is because of a direct protein-protein interaction. In response to DNA damage, PARP-1 de-localization and a reduction in bioluminescence resonance energy transfer signal because of the automodification reaction are observed, suggesting that the automodification reaction results in the disruption of the interaction between PARP-1 and Topo I. Because Topo I activity has been reported to be promoted by PARP-1, we then investigated the effect of the disruption of this interaction on Topo I activity, and we found that this disruption results in the reduction of Topo I activity. These results suggest that a function for the automodification reaction is to regulate the interaction between PARP-1 and Topo I, and consequently, the Topo I activity, in response to DNA damage.

Camptothecin-sensitive relaxation of supercoiled DNA by the topoisomerase I-like activity associated with poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerase-1 is a highly abundant nuclear enzyme implicated in transcription, DNA replication, and DNA repair through binding of nascent RNA and interactions with various factors. We found that purified fractions of recombinant human poly(ADP-ribose) polymerase-1 expressed in Escherichia coli possess yet another activity, a Mg(2+)-dependent DNA supercoil relaxation activity. Cleavage of recombinant poly(ADP-ribose) polymerase-1 by caspase-3, an apoptotic protease, reduced this activity, as did the removal of either of the two zinc finger motifs located in the N-terminal DNA-binding domain of poly(ADP-ribose) polymerase-1. In addition, this activity was separated from E. coli topoisomerase I by gel-filtration column chromatography, suggesting that this activity is specifically associated with poly(ADP-ribose) polymerase-1. Because this relaxation activity did not require ATP and was resistant to VP16, a topoisomerase II inhibitor, this activity is closer to that of topoisomerase I. However, the supercoiled DNA relaxation activity associated with poly(ADP-ribose) polymerase-1 is distinct from that of human or E. coli topoisomerase I, as this activity could not completely remove superhelical tensions from plasmid DNA. Thus, we referred to this activity as topoisomerase I-like activity. This Mg(2+)-dependent DNA supercoil relaxation activity was found to be sensitive to camptothecin, a mammalian topoisomerase I inhibitor.

Repair of single-strand DNA interruptions by redundant pathways and its implication in cellular sensitivity to DNA-damaging agents.

Single-strand DNA interruptions (SSIs) are produced during the process of base excision repair (BER). Through biochemical studies, two SSI repair subpathways have been identified: a pathway mediated by DNA polymerase beta (Pol beta) and DNA ligase III (Lig III), and a pathway mediated by DNA polymerase delta/epsilon (Pol delta/epsilon) and DNA ligase I (Lig I). In addition, the existence of another pathway, mediated by Pol beta and DNA Lig I, has been suggested. Although each pathway may play a unique role in cellular DNA damage response, the functional implications of SSI repair by these three pathways are not clearly understood. To obtain a better understanding of the functional relevance of SSI repair by these pathways, we investigated the involvement of each pathway by monitoring the utilization of DNA ligases in cell-free extracts. Our results suggest that the majority of SSIs produced during the repair of alkylated DNA bases are repaired by the pathway mediated by Pol beta and either Lig I or Lig III, although some SSIs are repaired by Pol delta/epsilon and Lig I. At a cellular level, we found that Lig III over-expression increased the resistance of cells to DNA-damaging agents, while Lig I over-expression had little effect. Thus, repair pathways mediated by Lig III may have a role in the regulation of cellular sensitivity to DNA-damaging agents.

Double-strand DNA break formation mediated by flap endonuclease-1.

Double-strand DNA breaks are the most lethal type of DNA damage induced by ionizing radiations. Previously, we reported that double-strand DNA breaks can be enzymatically produced from two DNA damages located on opposite DNA strands 18 or 30 base pairs apart in a cell-free double-strand DNA break formation assay (Vispé, S., and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392). In the assay that we developed, these two DNA damages are converted into single-strand interruptions by enzymes involved in base excision repair. We showed that these single-strand interruptions are converted into double-strand DNA breaks; however, it was not due to spontaneous denaturation of DNA. Thus, we proposed a model in which DNA polymerase delta/epsilon, by producing repair patches at single-strand interruptions, collide, resulting in double-strand DNA break formation. We tested the model and investigated whether other enzymes/factors are involved in double-strand DNA break formation. Here we report that, instead of DNA polymerase delta/epsilon, flap endonuclease-1 (FEN-1), an enzyme involved in base excision repair, is responsible for the formation of double-strand DNA break in the assay. Furthermore, by transfecting a flap endonuclease-1 expression construct into cells, thus altering their flap endonuclease-1 content, we found an increased number of double-strand DNA breaks after gamma-ray irradiation of these cells. These results suggest that flap endonuclease-1 acts as a double-strand DNA break formation factor. Because FEN-1 is an essential enzyme that plays its roles in DNA repair and DNA replication, DSBs may be produced in cells as by-products of the activity of FEN-1.