thapsigargin and 1-((3-5-dichloro)-2-6-dihydroxy-4-methoxyphenyl)-1-hexanone

We investigated the role of Atg1 in autophagic cell death (ACD) in a Dictyostelium monolayer model. The model is especially propitious, not only because of genetic tractability and absence of apoptosis machinery, but also because induction of ACD requires two successive exogenous signals, first the combination of starvation and cAMP, second the differentiation factor DIF-1. This enables one to analyze separately first-signal-induced autophagy and subsequent second-signal-induced ACD. We used mutants of atg1, a gene that plays an essential role in the initiation of autophagy. Upon starvation/cAMP, in contrast to parental cells, atg1 mutant cells showed irreversible lesions, clearly establishing a protective role for Atg1. Upon subsequent exposure to DIF-1 or to more ACD-specific second signals, starved parental cells progressed to ACD, but starved atg1 mutant cells did not, showing that Atg1 was required for ACD. Thus, in the same cells Atg1 was required in two apparently opposite ways, upon first-signaling for cell survival and upon second-signaling for ACD. Our findings strongly suggest that Atg1, thus presumably autophagy, protects the cells from starvation-induced cell death, allowing subsequent induction of ACD by the second signal. ACD is therefore not only "with" autophagy (since it showed signs of autophagy throughout), but is also "allowed by" autophagy. This does not exclude a role for autophagy also after second signaling. These results may account for discrepancies reported in the literature, encourage searches for second signals in different developmental models of ACD, and incite caution in autophagy-related therapeutic attempts.

Topics: Animals; Autophagy; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Dictyostelium; Enzyme Inhibitors; Hexanones; Ionophores; Models, Biological; Organisms, Genetically Modified; Protein Kinases; Signal Transduction; Thapsigargin

Differentiation-inducing factors (DIFs) are required for stalk cell formation in Dictyostelium discoideum. In the present study, in order to support our hypothesis that DIFs may function via increases in [Ca(2+)](c) and [H(+)](c), we investigated the combined effects of 5,5-dimethyl-2,4-oxazolidinedione (DMO, a [H(+)](c)-increasing agent), thapsigargin (Tg) and BHQ ([Ca(2+)](c)-increasing agents) on in vitro stalk cell formation in several strains. DMO, in combination with Tg or BHQ, induced stalk cell formation in a DIF-deficient mutant HM44. Although the rates of stalk cell induction by the drugs were low in the presence of cerulenin (an inhibitor of endogenous DIF production) in HM44 and V12M2 (a wild-type strain), the drugs succeeded in inducing sufficient stalk cell formation when a small amount of DIF-1 was supplied. Furthermore, co-addition of DMO, BHQ and a small amount of DIF-1 also induced sufficient stalk cell formation in AX-4 (an axenic strain) and HM1030 (dmtA(-)) but not in CT15 (dimA(-)). The drugs suppressed spore formation and promoted stalk cell formation in both HM18 (a sporogenous mutant) and 8-bromo-cAMP-stimulated V12M2. The present results suggest that DIFs function, at least in part, via increases in [Ca(2+)](c) and [H(+)](c) in D. discoideum.

Topics: Animals; Calcium; Cell Differentiation; Dictyostelium; Dimethadione; Enzyme Inhibitors; Hexanones; Hydrogen; Hydroquinones; Spores, Protozoan; Thapsigargin

The differentiation-inducing factor-1 (DIF-1) is a putative morphogen that induces stalk-cell formation in the lower eukaryote Dictyostelium discoideum. This molecule has been shown to inhibit cell growth and induce erythroid differentiation in human leukemia K562 cells. In the present study, to clarify the mechanism of the actions of DIF-1, we examined the effect of DIF-1 on Akt/protein kinase B (PKB) in K562 cells. Akt/PKB is a serine/threonine kinase that plays a pivotal role in the regulation of cell survival and differentiation in a variety of cells. A nonphosphorylated (inactive) form of Akt/PKB was ordinarily expressed in K562 cells. However, Akt/PKB was phosphorylated and potently activated within several hours of incubation with 5-30 microM DIF-1, and this activation was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase). Calcium-increasing agents thapsigargin and A23187 also activated Akt/PKB slightly, which was inhibited by wortmannin. By contrast, calcium-reducing agents TMB-8 and EGTA together with A23187 inhibited the DIF-1-induced activation of Akt/PKB. PMA (PKC activator) also activated Akt/PKB but this activation was not inhibited by wortmannin. DIF-1 exhibited no marked effect on the activation of PKCalpha, beta, and gamma, which were activated by PMA. These results indicate that DIF-1 activates Akt/PKB possibly via cytosolic calcium and subsequent activation of PI3-kinase and also that PMA activates Akt/PKB in a PI3-kinase-independent manner.

Topics: Androstadienes; Animals; Calcimycin; Calcium Channel Blockers; Cell Differentiation; Dictyostelium; Enzyme Activation; Enzyme Inhibitors; Erythrocytes; Gallic Acid; Hexanones; Humans; K562 Cells; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Structure-Activity Relationship; Thapsigargin; Wortmannin

During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+-sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from approximately 50 to 150 nM over a period of 8 h. The Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by approximately 1-2 h. The Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N'N'-tetra-acetic acid (BAPTA) efficiently inhibited the BHQ-induced [Ca2+]i increase and blocked DIF-induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2-]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Chelating Agents; Cytosol; Dictyostelium; Egtazic Acid; Enzyme Inhibitors; Extracellular Matrix Proteins; Fungal Proteins; Gene Expression Regulation, Developmental; Hexanones; Hydroquinones; Protozoan Proteins; Recombinant Fusion Proteins; Thapsigargin; Transcriptional Activation

A putative morphogen, called differentiation-inducing factor (DIF), is essential for stalk cell differentiation in the cellular slime mold, Dictyostelium discoideum. To investigate the relationship between the signal molecule (DIF) and the concentrations of cytoplasmic calcium ions and proton, we have examined the effects of thapsigargin (Tg) and 5,5-dimethyl-2,4-oxazolidinedione (DMO) on cell differentiation of a mutant strain HM44, which is defective in DIF production. Tg is a specific inhibitor of the Ca(2+)-ATPase present in endoplasmic and sarcoplasmic reticula, and raises the cytoplasmic calcium concentration. DMO is a reagent that decreases intracellular pH. When HM44 cells were incubated with Tg or DMO in the absence of DIF, a fraction of the cells was induced to stalk cells. If added together, these reagents induced stalk cell differentiation at high efficiency (70-80%), comparable to that attained with exogenous DIF. In the presence of the reagents, the efficiency was not much affected by lowering cell density, which suggests that the effect (stalk induction) of these reagents was not exerted through the stimulation of DIF production. Thus, these results indicate that a rise in cytoplasmic calcium and proton concentrations triggers stalk cell differentiation possibly by mimicking the roles of DIF.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cell Differentiation; Cytoplasm; Dictyostelium; Dimethadione; Dose-Response Relationship, Drug; Hexanones; Hydrogen-Ion Concentration; Kinetics; Models, Biological; Signal Transduction; Terpenes; Thapsigargin