Yuzuru Kubohara

Autophagy in Dictyostelium Genes and pathways, cell death and infection

The use of simple organisms to understand the molecular and cellular function of complex processes is instrumental for the rapid development of biomedical research. A remarkable example has been the discovery in S. cerevisiae of a group of proteins involved in the pathways of autophagy. Orthologues of these proteins have been identified in humans and experimental model organisms. Interestingly, some mammalian autophagy proteins do not seem to have homologues in yeast but are present in Dictyostelium, a social amoeba with two distinctive life styles, a unicellular stage in nutrient-rich conditions that differentiates upon starvation into a multicellular stage that depends on autophagy. This review focuses on the identification and annotation of the putative Dictyostelium autophagy genes and on the role of autophagy in development, cell death and infection by bacterial pathogens.

Differentiation-Inducing Factor-1 and -2 Function also as Modulators for Dictyostelium Chemotaxis

Background In the early stages of development of the cellular slime mold Dictyostelium discoideum, chemotaxis toward cAMP plays a pivotal role in organizing discrete cells into a multicellular structure. In this process, a series of signaling molecules, such as G-protein-coupled cell surface receptors for cAMP, phosphatidylinositol metabolites, and cyclic nucleotides, function as the signal transducers for controlling dynamics of cytoskeleton. Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2) were originally identified as the factors (chlorinated alkylphenones) that induce Dictyostelium stalk cell differentiation, but it remained unknown whether the DIFs had any other physiologic functions. Methodology/Principal Findings To further elucidate the functions of DIFs, in the present study we investigated their effects on chemotaxis under various conditions. Quite interestingly, in shallow cAMP gradients, DIF-1 suppressed chemotaxis whereas DIF-2 promoted it greatly. Analyses with various mutants revealed that DIF-1 may inhibit chemotaxis, at least in part, via GbpB (a phosphodiesterase) and a decrease in the intracellular cGMP concentration ([cGMP]i). DIF-2, by contrast, may enhance chemotaxis, at least in part, via RegA (another phosphodiesterase) and an increase in [cGMP]i. Using null mutants for DimA and DimB, the transcription factors that are required for DIF-dependent prestalk differentiation, we also showed that the mechanisms for the modulation of chemotaxis by DIFs differ from those for the induction of cell differentiation by DIFs, at least in part. Conclusions/Significance Our findings indicate that DIF-1 and DIF-2 function as negative and positive modulators for Dictyostelium chemotaxis, respectively. To our knowledge, this is the first report in any organism of physiologic modulators (small molecules) for chemotaxis having differentiation-inducing activity.

Autophagic or necrotic cell death triggered by distinct motifs of the differentiation factor DIF-1

Autophagic or necrotic cell death (ACD and NCD, respectively), studied in the model organism Dictyostelium which offers unique advantages, require triggering by the same differentiation-inducing factor DIF-1. To initiate these two types of cell death, does DIF-1 act through only one or through two distinct recognition structures? Such distinct structures may recognize distinct motifs of DIF-1. To test this albeit indirectly, DIF-1 was modified at one or two of several positions, and the corresponding derivatives were tested for their abilities to induce ACD or NCD. The results strongly indicated that distinct biochemical motifs of DIF-1 were required to trigger ACD or NCD, and that these motifs were separately recognized at the onset of ACD or NCD. In addition, both ACD and NCD were induced more efficiently by DIF-1 than by either its precursors or its immediate catabolite. These results showed an unexpected relation between a differentiation factor, the cellular structures that recognize it, the cell death types it can trigger and the metabolic state of the cell. The latter seems to guide the choice of the signaling pathway to cell death, which in turn imposes the cell death type and the recognition pattern of the differentiation factor.

Regulation of IL-2 production in Jurkat cells by Dictyostelium-derived factors

AIMSnDifferentiation-inducing factors (DIFs) are chlorinated alkylphenones found in the cellular slime mold Dictyostelium discoideum. DIF derivatives exhibit antiproliferative activities and promote glucose consumption in mammalian cells in vitro. In this study, we assessed the ability of DIFs to regulate the immune system and investigated their mechanisms of action.nnnMAIN METHODSnWe examined the effects of 30 DIF derivatives on concanavalin A-induced IL-2 production (CIIP) in Jurkat T-cells. We also examined the effects of some DIF derivatives on the activity of AP-1 (activator protein-1), NFAT (nuclear factor of activated T-cells), and NFkappaB (nuclear factor kappa B), which are transcription factors required for CIIP.nnnKEY FINDINGSnOf the derivatives tested, some compounds suppressed CIIP as well as the known immunosuppressants cyclosporine A and FK506. A reporter gene assay revealed that 4 DIF derivatives tested suppressed CIIP, at least in part, by inhibiting the activity of AP-1, NFAT, and/or NFkappaB. Unlike cyclosporine A and FK506, the DIF derivatives had little effect on concanavalin A-induced interferon-gamma production in Jurkat cells.nnnSIGNIFICANCEnThe present results suggest that DIF derivatives could be developed as novel immunosuppressive drugs.

Mitochondria Are the Target Organelle of Differentiation-Inducing Factor-3, an Anti-Tumor Agent Isolated from Dictyostelium Discoideum

Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3 (Bu-DIF-3) are potent anti-tumor agents. However, the precise mechanisms underlying the actions of DIF-3 remain to be elucidated. In this study, we synthesized a green fluorescent derivative of DIF-3, BODIPY-DIF-3, and a control fluorescent compound, Bu-BODIPY (butyl-BODIPY), and investigated how DIF-like molecules behave in human cervical cancer HeLa cells by using both fluorescence and electron microscopy. BODIPY-DIF-3 at 5–20 µ M suppressed cell growth in a dose-dependent manner, whereas Bu-BODIPY had minimal effect on cell growth. When cells were incubated with BODIPY-DIF-3 at 20 µM, it penetrated cell membranes within 0.5 h and localized mainly in mitochondria, while Bu-BODIPY did not stain the cells. Exposure of cells for 1–3 days to DIF-3, Bu-DIF-3, BODIPY-DIF-3, or CCCP (a mitochondrial uncoupler) induced substantial mitochondrial swelling, suppressing cell growth. When added to isolated mitochondria, DIF-3, Bu-DIF-3, and BOIDPY-DIF-3, like CCCP, dose-dependently promoted the rate of oxygen consumption, but Bu-BODIPY did not. Our results suggest that these bioactive DIF-like molecules suppress cell growth, at least in part, by disturbing mitochondrial activity. This is the first report showing the cellular localization and behavior of DIF-like molecules in mammalian tumor cells.

Derivatives of Dictyostelium differentiation-inducing factors promote mitogen-activated IL-2 production via AP-1 in Jurkat cells

AIMSnDifferentiation-inducing factors (DIFs) are chlorinated alkylphenones found in the cellular slime mold Dictyostelium discoideum. DIF derivatives exhibit antiproliferative activities and promote glucose consumption in mammalian cells in vitro. Here, we assessed the ability of DIFs to regulate the immune system in a mammalian cell-line and investigated their mechanisms of action.nnnMAIN METHODSnWe examined the effects of 30 DIF derivatives on concanavalin A-induced interleukin-2 (IL-2) production (CIIP) in Jurkat T-cells. We also examined the effects of these DIF derivatives on the activity of three transcription factors required for CIIP: namely, activator protein-1 (AP-1), nuclear factor of activated T-cells (NFAT), and nuclear factor kappa B (NFκB).nnnKEY FINDINGSnA reporter gene assay suggested that 2 DIF derivatives, termed DIF-1(+1) and DIF-3(3M), significantly promoted CIIP in Jurkat cells, at least in part, by enhancing the activity of AP-1. These 2 DIF derivatives had no significant effect on concanavalin A-induced interferon-γ production.nnnSIGNIFICANCEnThe results suggest that DIF derivatives could be developed as novel drugs for the activation of IL-2 production and resultant stimulation of the immune system.

Novel chlorinated dibenzofurans isolated from the cellular slime mold, Polysphondylium filamentosum, and their biological activities.

Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster.

Br-DIF-1 accelerates dimethyl sulphoxide-induced differentiation of P19CL6 embryonic carcinoma cells into cardiomyocytes

BACKGROUND AND PURPOSE Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte‐like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation‐inducing factor‐1 (DIF‐1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells.

Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acid-stimulated migration of murine osteosarcoma LM8 cells.

Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1),xa0-2, andxa0-3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5-20xa0μM) dose-dependently suppressed LPA-induced cell migration with associated IC50 values of 5.5, 4.6, and 4.2xa0μM, respectively. On the other hand, the IC50 values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0xa0μM, respectively, in LM8 cells, and >20, 14.8, and 4.3xa0μM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis.

Properties of a non-bioactive fluorescent derivative of differentiation-inducing factor-3, an anti-tumor agent found in Dictyostelium discoideum.

ABSTRACT Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives, such as butoxy-DIF-3 (Bu-DIF-3), are potent anti-tumor agents. To investigate the activity of DIF-like molecules in tumor cells, we recently synthesized a green fluorescent DIF-3 derivative, BODIPY-DIF-3G, and analyzed its bioactivity and cellular localization. In this study, we synthesized a red (orange) fluorescent DIF-3 derivative, BODIPY-DIF-3R, and compared the cellular localization and bioactivities of the two BODIPY-DIF-3s in HeLa human cervical cancer cells. Both fluorescent compounds penetrated the extracellular membrane within 0.5u2005h and localized mainly to the mitochondria. In formalin-fixed cells, the two BODIPY-DIF-3s also localized to the mitochondria, indicating that the BODIPY-DIF-3s were incorporated into mitochondria independently of the mitochondrial membrane potential. After treatment for 3 days, BODIPY-DIF-3G, but not BODIPY-DIF-3R, induced mitochondrial swelling and suppressed cell proliferation. Interestingly, the swollen mitochondria were stainable with BODIPY-DIF-3G but not with BODIPY-DIF-3R. When added to isolated mitochondria in vitro, BODIPY-DIF-3G increased dose-dependently the rate of O2 consumption, but BODIPY-DIF-3R did not. These results suggest that the bioactive BODIPY-DIF-3G suppresses cell proliferation, at least in part, by altering mitochondrial activity, whereas the non-bioactive BODIPY-DIF-3R localizes to the mitochondria but does not affect mitochondrial activity or cell proliferation.