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

The DIFs are a family of secreted chlorinated molecules that control cell fate during development of Dictyostelium cells in culture and probably during normal development too. They induce stalk cell differentiation and suppress spore cell formation. The biosynthetic and inactivation pathways of DIF-1 (the major bioactivity) have been worked out. DIF-1 is probably synthesised in prespore cells and inactivated in prestalk cells, by dechlorination. Thus, each cell type tends to alter DIF-1 level so as to favour differentiation of the other cell type. This relationship leads to a model for cell-type proportioning during normal development.

Topics: Animals; Cell Differentiation; Cell Division; Dictyostelium; Hexanones; Lyases; Oxidoreductases; Pentanones; Signal Transduction

Prestalk cell differentiation in Dictyostelium is induced by DIF and two DIF-induced genes, ecmA and ecmB, have revealed the existence of multiple prestalk and stalk cell sub-types. These different sub-types are defined by the pattern of expression of subfragments derived from the ecmA and ecmB promoters. These markers have been utilised in three ways; for fate mapping in vivo, to investigate the molecular mechanisms underlying DIF signalling and to explore the relative requirement for DIF and other signalling molecules for prestalk and stalk cell differentiation in vitro. The heterogeneity of the prestalk and stalk populations seems to be reflected in differences in the cell signalling pathways that they utilise.

Topics: Animals; Cell Differentiation; Dictyostelium; Genes, Protozoan; Hexanones; Lyases; Oxidoreductases; Pentanones; Protozoan Proteins; Signal Transduction

Topics: Cell Differentiation; Chemotaxis; Cyclic AMP; Dictyostelium; Fungal Proteins; Gene Expression Regulation, Fungal; Genes, Fungal; Hexanones; Pentanones; ras Proteins; Signal Transduction; Transcription Factors

Topics: Adenosine; Ammonia; Cyclic AMP; Dictyostelium; Hexanones; Morphogenesis; Pentanones

We found novel development rescuing factors (DRFs) secreted from developing Dictyostelium cells, by using a mutant (erkB-) which is missing MAP-kinase ERK2 as a test strain for bioassay. The mutant erkB- fails to undergo multicellular morphogenesis due to impaired cAMP signaling. However, such developmental defect can be restored by the presence of low-molecular weight DRFs that are secreted from developing wild-type cells. We previously showed that DIF-1 (Differentiation-Inducing Factor 1 for stalk cells) possesses this activity, indicating a newly discovered role of DIF-1. Surprisingly, however, the mutant dmtA-, which is incapable of DIF-1 synthesis still exerts a strong inducing activity of the multicellular morphogenesis of erkB-. After analysis of HPLC fractions of conditioned media prepared from both wild type Ax2 and dmtA- strains revealed that both strains secrete at least two novel DRF activities with DIF-like mobility. However, these activities were not derived from other DIFs such as DIF-2 and DIF-3. Identification of these DRFs found in this study would provide insight into the mechanism by which the development of the erkB- mutant is restored and how these factors act in the normal development of Dictyostelium.

Topics: Animals; Cell Differentiation; Chromatography, High Pressure Liquid; Dictyostelium; Gene Expression Regulation, Developmental; Hexanones; Mitogen-Activated Protein Kinase 1; Morphogenesis; Pentanones; Signal Transduction

Cell fate in Dictyostelium development depends on intrinsic differences between cells, dating from their growth period, and on cell interactions occurring during development. We have sought for a mechanism linking these two influences on cell fate. First, we confirmed earlier work showing that the vegetative differences are biases, not commitments, since cells that are stalky-biased when developed with one partner are sporey with another. Then we tested the idea that these biases operate by modulating the sensitivity of cells to the signals controlling cell fate during development. Cells grown without glucose are stalky-biased when developed with cells grown with glucose. We find, using monolayer culture conditions, that they are more sensitive to each of the stalk-inducing signals, DIFs 1-3. Mixing experiments show that this bias is a cell-intrinsic property. Cells initiating development early in the cell cycle are stalky compared to those initiating development later in the cycle. Likewise, they are more sensitive to DIF-1. Assays of standard markers for prestalk and prespore cell differentiation reveal similar differences in DIF-1 sensitivity between biased cells; DIF-1 dechlorinase (an early prestalk cell marker enzyme) behaves in a consistent manner. We propose that cell-fate biases are manifest as differences in sensitivity to DIF.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Biomarkers; Cell Cycle; Cell Differentiation; Cell Lineage; Dictyostelium; Enzyme Induction; Gene Expression Regulation, Developmental; Glucose; Green Fluorescent Proteins; Hexanones; Luminescent Proteins; Lyases; Oxidoreductases; Pentanones; Signal Transduction; Spores

DIF-1 (differentiation-inducing factor-1; 1-(3,5-dichloro-2, 6-dihydroxy-4-methoxyphenyl)hexan-1-one) is a putative morphogen that induces stalk cell formation in the cellular slime mold, Dictyostelium discoideum. It has been previously reported that DIF-1 exhibits anti-tumor activity in mammalian cells. In this study, we examined the effects of six DIF analogues on DNA synthesis, cell growth, erythroid differentiation, and cytosolic free calcium concentration ([Ca2+]i) in human leukemia K562 cells. The DIF analogues used here were DIF-1, DIF-2 (which has pentanone in place of hexanone), DIF-3 (dechlorinated form of DIF-1), 2-MIDIF-1 (2-methoxy isomer of DIF-1), DMPH (dechlorinated form of DIF-3), and THPH (4-hydroxy substitution of DMPH). DIF-3 proved to be the most potent anti-leukemic agent among them, and the order of potency for causing growth inhibition, erythroid induction, and increases in [Ca2+]iTHPH in all the categories tested. The present results suggest new routes for the development of more potent and effective anti-tumor agents.

Topics: Animals; Antineoplastic Agents; Calcium; Cell Count; Cell Differentiation; Cell Division; Dictyostelium; DNA; Dose-Response Relationship, Drug; Hexanones; Humans; K562 Cells; Leukemia; Pentanones; Phenols; Resorcinols

Stalk cell differentiation in Dictyostelium can be induced by the differentiation-inducing factor, DIF, or by conditions that decrease intracellular pH (pHi). We have investigated whether cytoplasmic acidification acts directly to induce expression of pDd56 and pDd63, two DIF-regulated genes, specifically expressed in prestalk cells. The weak base methylamine, which increases pHi, inhibits DIF-induced transcription. The weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO), which decreases pHi, stimulates DIF-induction of the two prestalk genes. After relatively long incubation periods, DMO also induces a low level of prestalk gene expression in the absence of added DIF. However, unlike DIF-mediated induction, the apparent DMO-mediated induction decreases to undetectable levels when the cell density is reduced from 10(7) to 10(5) cells/ml. This indicates that DMO does not itself induce gene expression, but acts to enhance the effects of an autonomously secreted stalk-inducing factor, presumably DIF. These results suggest that the effects of DIF on gene expression are regulated by intracellular pH, but do not support a role for protons as direct intermediates in the DIF signal transduction pathway.

Topics: Cyclic AMP; Dictyostelium; Dimethadione; Gene Expression Regulation, Fungal; Hexanones; Hydrogen-Ion Concentration; Methylamines; Pentanones; RNA, Messenger

The DIFs are unusual, chlorinated molecules which induce stalk cell differentiation during the later, multicellular phase of Dictyostelium development. Here we provide evidence that one or more DIFs have a role during early development, when small amounts are known to be made. Initial indications came from an optical technique which detects changes in shape or cohesion of cells in suspension (Gerisch and Hess, PNAS 71, 2118, 1974). After a period of optical inactivity at the start of development, cell suspensions normally produce spontaneous spike-shaped light-scattering oscillations synchronised by oscillations in extracellular cAMP levels, followed by sinusoidal oscillations where the synchroniser is not known. DIFs 1 and 2 produce optical responses from cells at all these early stages of development. The phase of both spiked and sinusoidal oscillations can be shifted, indicating an effect on the oscillator in each case. We find further: (1) cAMP oscillations and cAMP relay during spiked oscillations are transiently inhibited by DIF-1. (2) DIF-1 causes a transient decrease in cellular cGMP levels in cells taken before oscillations commence and likewise inhibits the cGMP response to a cAMP stimulus in cells taken later in development. Cytoskeletal organization and hence cell shape might be affected by DIF-1 by this indirect route. (3) The effects of DIF-1 are transient, even though it is essentially stable in the cell suspension. Cells somehow adapt to DIF-1. (4) The effects are chemically specific: DIF-1 and DIF-2 are active at 10(-7) to 10(-8) M, with DIF-2 being the more active, whereas related compounds have little or no activity at 10(-6) M. These results indicate that cells are responsive to DIFs 1 and 2 from the start of development and suggest a wider role for the DIFs. This role might involve effects on cAMP signalling and on intracellular second messengers.

Topics: Cell Adhesion; Cell Aggregation; Cyclic AMP; Cyclic GMP; Dictyostelium; Dose-Response Relationship, Drug; Hexanones; Ketones; Pentanones

In Dictyostelium discoideum stalk cell formation is induced by cyclic AMP and differentiation-inducing factor (DIF) when cells are plated in in vitro monolayers (Kay et al., 1979, Differentiation 13: 7-14). The in vivo developmental stages at which cells became independent of these factors were determined. Independence was defined as the stage at which dispersed cells no longer required the factors for stalk cell formation in low density monolayers. Cyclic AMP independent cells were first detected at around 12 hr of development, a time that corresponds to the transition between the tipped aggregate and the first finger stages. In contrast cells did not become independent of DIF until late culmination. The prestalk cell-specific isozyme acid phosphatase II and a stalk cell-specific 41,000 Mr antigen (ST 41) were expressed during differentiation in low density monolayers in the presence of both cyclic AMP and DIF, but neither component was expressed in the presence of cyclic AMP alone. This result implies that DIF is essential for both prestalk and stalk cell formation. The two components were expressed within 2 hr of each other during differentiation in vitro, whereas during development in vivo acid phosphatase II was first detected at the first finger stage and ST 41 was first detected during late culmination, 8-12 hr later. These contrasting results suggest that the conversion of prestalk cells to stalk cells is unrestrained in monolayers, following directly after prestalk cell induction, but restrained in vivo until the culmination stage. This interpretation is consistent with the finding that cells become independent of DIF early during in vitro differentiation (A. Sobolewski, N. Neave, and G. Weeks, 1983, Differentiation 25, 93-100), but do not become independent of DIF until the culmination stage when differentiating in vivo.

Topics: Acid Phosphatase; Antigens, Fungal; Cell Differentiation; Cyclic AMP; Dictyostelium; Hexanones; Isoenzymes; Ketones; Morphogenesis; Pentanones; Time Factors

Previous work has led to the identification of a novel class of effector molecules [DIFs (differentiation-inducing factors) 1-3] released from the slime mould Dictyostelium discoideum. These substances induce stalk-cell differentiation in Dictyostelium discoideum and are thought to act as morphogens in the generation of the prestalk/prespore pattern during development. The DIFs are phenylalkan-1-ones, with chloro, hydroxy and methoxy substitution on the benzene ring. DIFs 1-3 and a number of their analogues have been synthesized by using a simple two-step procedure, and each analogue has been characterized by m.s., u.v. and n.m.r. spectroscopy. The crystal structure of synthetic DIF-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one, was investigated. The specific biological activity of each analogue was determined in a bioassay, where isolated Dictyostelium amoebae are induced to differentiate into stalk cells. The major biologically active substance, DIF-1, caused 50% stalk-cell differentiation at 1.8 x 10(-10) M; the C4 alkyl homologue (DIF-2) and C6 homologue possessed 40 and 16% of the activity of DIF-1 respectively. Further increase or decrease in the alkyl chain length resulted in a marked decrease in specific activity. The pattern of substitution on the benzene ring is a major determinant of bioactivity, since the specific activities of the 2,4-dihydroxy-6-methoxy and trihydroxy analogues were less than 1% of that of DIF-1. Substitution of bromine in DIF-1 had little effect on bioactivity; in contrast the activity of monochloro-DIF-1 (DIF-3) was diminished. There was no evidence for antagonism or synergy between DIF-1 and any of its analogues. This series of analogues will facilitate further studies in the biological effects and mode of action of DIF-1.

Topics: Cell Differentiation; Chromatography, High Pressure Liquid; Dictyostelium; Hexanones; Ketones; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Molecular; Pentanones