Stalk Cell Formation Research Articles

Effects of differentiation-inducing factors of Dictyostelium discoideum on human leukemia K562 cells: DIF-3 is the most potent anti-leukemic agent

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 ([Ca 2+] 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 [Ca 2+] i was established as DIF-3>DIF-1>DIF-2>DMPH>2-MIDIF-1>THPH in all the categories tested. The present results suggest new routes for the development of more potent and effective anti-tumor agents.

Mosaic analysis in the drosophila ovary reveals a common hedgehog-inducible precursor stage for stalk and polar cells.

The fates of two small subgroups of the ovarian follicle cells appear to be linked: mutations in Notch, Delta, fs(1)Yb, or hedgehog cause simultaneous defects in the specification of stalk cells and polar cells. Both of these subgroups are determined in the germarium, and both cease division early in oogenesis. To test the possibility that these subgroups are related by lineage, we generated dominantly marked mitotic clones in ovaries. Small, restricted clones in stalk cells and polar cells were found adjacent to each other at a frequency much too high to be explained by independent induction. We therefore propose a model in which stalk cells and polar cells are derived from a precursor population that is distinct from the precursors for other follicle cells. We support and extend this model by characterization of mutants that affect stalk and polar cell formation. We find that ectopic expression of Hedgehog can induce both polar and stalk cell fate, presumably by acting on the precursor stage. In contrast, we find that stall affects neither the induction of the precursors nor the decision between the stalk cell and polar cell fate but, rather, some later differentiation step of stalk cells. In addition, we show that ectopic polar and stalk cells disturb the anterior-posterior polarity of the underlying oocyte.

Production and activity of spore differentiation factors (SDFs) in Dictyostelium.

SDF-1 and SDF-2 are peptides that promote terminal spore differentiation under submerged conditions. The present study shows that they accumulate differentially and are released during the development of wild-type cells and can promote spore formation in cells disaggregated from wild-type culminants. SDF-1 accumulates during the slug stage and is released in a single burst at the onset of culmination while SDF-2 accumulates during early culmination and is released in a single burst from mid-culminants. The effects of SDF-1 and SDF-2 on stalk cell formation in cell monolayers were investigated. SDF-1 by itself induces stalk cell formation in some strains and also synergizes with the stalk-cell-inducing factor, DIF-1. cAMP has an inhibitory effect on stalk cell formation when either DIF-1 or SDF-1 are present on their own but is almost not inhibitory when both are present. SDF-2 alone does not induce stalk cell formation and appears to inhibit the response to DIF-1. At the same time, it increases the extent of vacuolization of the stalk cells that are produced. We propose that the release of SDF-1 and then of SDF-2 may mark irreversible steps in the developmental programme associated, respectively, with culmination and spore maturation.

Effects of DIF-1, an Anti-Tumor Agent Isolated from Dictyostelium discoideum, on Rat Gastric Mucosal RGM-1 and Leptomeningeal Cells

DIF-1 is a putative morphogen that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. We have previously discovered that DIF-1 suppresses cell growth and induces cell differentiation in vitro in some tumor cells, and also that relatively low concentrations of DIF-1 promote retinoic acid-induced cell differentiation in the human myeloid leukemia HL-60 cells. In this study, to verify cell biological and therapeutic potential of DIF-1, we have examined whether and how DIF-1 affects normal mammalian cells in vitro, using rat leptomeningeal (RLM) cells and rat gastric mucosal RGM-1 cells. In growing phase of both cells, DIF-1 at 5–40 μM suppressed cell growth in a dose-dependent manner. High concentrations (15–40 μM) of DIF-1 were toxic to the growing cells so that the cells showed unusual morphology, but many of them were still alive even at Day 3–4. Withdrawal of DIF-1 allowed the cells to grow. In confluent phase of the cells, DIF-1 at more than 15 μM promoted medium acidification that resulted in cell death, but DIF-1 itself did not markedly affect cell viability for 3 days. DIF-1 increased [Ca2 ]i in RLM cells but did not affect β-trace secretion (an index of cell function in RLM cells). A low concentration (5 μM) of DIF-1 in combination with retinoic acid (0.1 μM) showed no marked effects on cell growth, cell viability, cell morphology and β-trace secretion in RLM cells. The present results indicate that DIF-1 may be utilized as a tool for cell biology. Also, since these concentrations of DIF-1 kill some tumor cells within 2–3 days, the present results suggest that DIF-1 might be utilized in the treatment of some sorts of cancer with a bearable degree of side-effects.

Role of PKA in the timing of developmental events in Dictyostelium cells.

The cyclic AMP (cAMP)-dependent protein kinase, PKA, is dispensable for growth of Dictyostelium cells but plays a variety of crucial roles in development. The catalytic subunit of PKA is inhibited when associated with its regulatory subunit but is activated when cAMP binds to the regulatory subunit. Deletion of pkaR or overexpression of the gene encoding the catalytic subunit, pkaC, results in constitutive activity. Development is independent of cAMP in strains carrying these genetic alterations and proceeds rapidly to the formation of both spores and stalk cells. However, morphogenesis is aberrant in these mutants. In the wild type, PKA activity functions in a circuit that can spontaneously generate pulses of cAMP necessary for long-range aggregation. It is also essential for transcriptional activation of both prespore and prestalk genes during the slug stage. During culmination, PKA functions in both prespore and prestalk cells to regulate the relative timing of terminal differentiation. A positive feedback loop results in the rapid release of a signal peptide, SDF-2, when prestalk cells are exposed to low levels of SDF-2. The signal transduction pathway that mediates the response to SDF-2 in both prestalk and prespore cells involves the two-component system of DhkA and RegA. When the cAMP phosphodiesterase RegA is inhibited, cAMP accumulates and activates PKA, leading to vacuolation of stalk cells and encapsulation of spores. These studies indicate that multiple inputs regulate PKA activity to control the relative timing of differentiations in Dictyostelium.

Evaluation of the Teratogenic Potential of Valproic Acid Analogues in Transgenic Dictyostelium discoideum Strains

Very early during the development of new pharmaceuticals toxicological tests are most important. In addition to acute and chronic toxicity tests, it is crucial to estimate the teratogenic potential of promising drugs. We established a simple biological test system based on the cellular slime mold Dictyostelium discoideum. Under certain environmental conditions single cells of D. discoideum aggregate and undergo a relatively simple cell differentiation program, leading to the formation of stalk and spore cells. Transgenic D. discoideum strains carrying the bacterial β-galactosidase gene under the control of various developmentally regulated D. discoideum promoters were shown to be useful tools to test the teratogenic potential of valproic acid (VPA). This study describes the effects of the VPA analogues S-4-yn-VPA, R-4-yn-VPA, and 2-ethyl-4-pentynoic acid on the D. discoideum developmental system. The presence of S-4-yn-VPA during D. discoideum development resulted in a strong inhibition of spore cell differentiation, whereas stalk cell formation was less affected. The enantiomer R-4-yn-VPA as well as 2-ethyl-4-pentynoic acid had only moderate effects on D. discoideum development. The above results are consistent with data obtained in mammalian teratogenicity assays, and suggest that D. discoideum development should be investigated with a number of additional substances to provide a simple alternative for high throughput screenings of new drugs.

Efficient induction by DIF-1 and 8-bromo cyclic AMP of prespore-to-stalk conversion in Dictyostelium discoideum

We have examined the in vitro effects of DIF-1 (differentiation-inducing factor-1), DIF-2, cAMP, and/or 8-bromo cAMP (Br-cAMP) on prespore cells isolated from normally formed slugs of the cellular slime mold, Dictyostelium discoideum (strain NC4). Without these factors, the isolated cells remained amoeboid at 24 hr of incubation (at 5 × 10 3 cells/cm 2). DIF-1 (100 nM) poorly induced the prespore cells to differentiate into stalk cells (< 15%), and DIF-2 (100 nM) was a less effective inducer of stalk cells (< 1%). Cyclic AMP (200 μM) itself induced neither stalk- nor spore-formation but was inhibitory to the DIF-1-induced stalk cell formation. Br-cAMP (10 mM) alone induced prespore-to-spore maturation. It was found that prespore cells were converted to stalk cells efficiently (> 85%) in the presence of both Br-cAMP and DIF-1.

Rap1 Overexpression Reveals That Activated RasD Induces Separable Defects duringDictyosteliumDevelopment

One of theDictyostelium rasgenes,rasD,is expressed preferentially in prestalk cells at the slug stage of development and overexpression of this gene containing a G12T activating mutation causes the formation of aberrant multitipped aggregates that are blocked from further development (Reymondet al.,1986,Nature,323, 340–343). The ability of theDictyostelium rap1gene to suppress this abnormal developmental phenotype was investigated. Therap1gene and G12V activated and G10V negative mutant forms of therap1gene were independently linked to therasDpromoter and each construct used to transform M1, aDictyosteliumcell line expressing RasD[G12T]. Transformants of M1 that expressed Rap1 or Rap1[G12V] protein still formed multitipped aggregates, but most tips were able to complete development and form fruiting bodies. Cell lines showing this modified phenotype were designated ME (multitipped escape). Therap1[G10V] construct did not modify the M1 phenotype. These data suggest that overexpression of RasD[G12T] has two effects, the formation of a multitipped aggregate and a block in subsequent differentiation and that the expression of Rap1 or Rap1[G12V] reverses only the latter. Differentiation of ME cells in low density monolayers showed the identical low level of stalk and spore cell formation seen for M1 cells under the same conditions. Thus the cell autonomous defect in monolayer differentiation induced in the M1 strain was not corrected in the ME strain. Cell type-specific gene expression during the development of M1 cells is dramatically altered: prestalk cell-specific gene expression is greatly enhanced, whereas prespore-specific gene expression is almost suppressed (Louiset al.,1997,Mol. Biol. Cell,8, 303–312). During the development of ME cells,ecmA mRNA levels were restored to those seen for Ax3, andtagB mRNA levels were also markedly reduced, although not to Ax3 levels.cotCexpression in ME cells was enhanced severalfold relative to M1, although levels were still lower than those observed during the development of Ax3. The low expression ofcar1mRNA during early development of the M1 strain remained low during the development of ME cells. These data are consistent with the idea that the expression of RasD[G12T] affects two independent and temporally separated events and that only the later defect is reversed byrap1.

DIF-1, Putative Morphogen ofD. discoideum,Suppresses Cell Growth and Promotes Retinoic Acid-Induced Cell Differentiation in HL-60

Differentiation-inducing factor-1 (DIF-1) is a putative morphogen that induces stalk cell formation in the cellular slime moldDictyostelium discoideum.In this study, we have examined the effects of DIF-1 on the human leukemia HL-60 cells. DIF-1 at 10–40 μM suppressed cell growth in a dose-dependent manner, and approximately 50% growth inhibition was attained with 15–20 μM DIF-1. FACS analysis of cell-cycle phase distribution using propidium iodide revealed that many cells were accumulated in the G1 phase after treatment with 15–20 μM DIF-1. These concentrations of DIF-1 also raised [Ca2+]iin a dose-dependent manner irrespective of the presence of extracellular Ca2+, indicating that DIF-1 elicited Ca2+-release from some intracellular Ca2+store(s). Most importantly, relatively low concentrations of DIF-1 (1–5 μM) were found to promote retinoic acid-induced cell differentiation. The present results indicate that DIF-1 may be a useful tool for the analysis of myeloid cell differentiation and have therapeutic potential in the treatment of human myeloid leukemia.

Expression of an activated rasD gene changes cell fate decisions during Dictyostelium development.

It has been previously demonstrated that the expression of an activated rasD gene in wild-type Dictyostelium cells results in formation of aggregates with multitips, instead of the normal single tips, and a block in further development. In an attempt to better understand the role of activated RasD development, we examined cell-type-specific gene expression in a strain stably expressing high levels of RasD[G12T]. We found that the expression of prestalk cell-specific genes ecmA and tagB was markedly enhanced, whereas the expression of the prespore cell-specific gene cotC was reduced to very low levels. When the fate of cells in the multitipped aggregate was monitored with an ecmA/lacZ fusion, it appeared that most of the cells eventually adopted prestalk gene expression characteristics. When mixtures of the [G12T]rasD cells and Ax3 cells were induced to differentiate, chimeric pseudoplasmodia were not formed. Thus, although the [G12T]rasD transformant had a marked propensity to form prestalk cells, it could not supply the prestalk cell population when mixed with wild-type cells. Both stalk and spore cell formation occurred in low cell density monolayers of the [G12T]rasD strain, suggesting that at least part of the inhibition of stalk and spore formation during multicellular development involved inhibitory cell interactions within the cell mass. Models for the possible role of rasD in development are discussed.

A role for calcineurin in Dictyostelium discoideum development

We have used the immunosuppressants cyclosporin A and FK506 to investigate the involvement of the Ca 2+/CaM-dependent protein phosphatase calcineurin in Dictyostelium discoideum development. We found that CsA had little effect on cell growth, or on the aggregation of developing amoebae, suggesting that calcineurin does not play a significant role at these stages of the D. discoideum life cycle. In contrast, when cells were allowed to differentiate under buffer in the presence of cAMP, addition of CsA and FK506 strongly inhibited stalk cell formation in the wild-type and spore formation in a sporogenous derivative of D. discoideum strain V12. These agents also reduced the expression of prestalk- and prespore-specific transcripts in both strains. These results indicate a requirement for calcineurin activity in both pathways of cell differentiation. In addition, time-course experiments suggest that calcineurin is required early in the differentiation processes, but that the maturation of the two cell types is resistant to calcineurin inhibition. We also found that CsA and FK506 were unable to affect spore formation in rapidly developing/sporogenous rdeC mutants of strain NC4, showing that constitutive cAMP-dependent protein kinase activity renders the spore pathway resistant to calcineurin inhibition.

Cyclic AMP dependent protein kinase and prestalk-cell gene expression inDictyostelium

Cyclic AMP-dependent protein kinase (PKA) is required for aggregation of starved amoebae during development of Dictyostelium discoideum as well as for prespore gene expression and for maturation of stalk cells and spores. It has not been clear until now whether it plays any role in the stalk pathway prior to the final maturation of stalk cells. We have examined stalk cell formation and prestalk cell gene expression in a mutant of Dictyostelium that has an inactive PKA regulatory subunit and therefore has unrestrained catalytic activity (Nature 356 (1992) 171–172). We show that when mutant amoebae are incubated under buffer in the absence of cAMP they accumulate transcripts of the prestalk-cell-specific ecmA gene and form stalk cells, whereas the parental cells neither accumulate ecmA transcripts nor form stalk cells. These findings indicate that constitutive PKA activity renders cells able to express prestalk-cell-specific genes under conditions where wild-type cells cannot do so, and hence that PKA is probably implicated in prestalk gene expression during normal development.

Cyclic AMP dependent protein kinase and prestalk-cell gene expression in Dictyostelium

Cyclic AMP-dependent protein kinase (PKA) is required for aggregation of starved amoebae during development of Dictyostelium discoideum as well as for prespore gene expression and for maturation of stalk cells and spores. It has not been clear until now whether it plays any role in the stalk pathway prior to the final maturation of stalk cells. We have examined stalk cell formation and prestalk cell gene expression in a mutant of Dictyostelium that has an inactive PKA regulatory subunit and therefore has unrestrained catalytic activity (Nature 356 (1992) 171–172). We show that when mutant amoebae are incubated under buffer in the absence of cAMP they accumulate transcripts of the prestalk-cell-specific ecmA gene and form stalk cells, whereas the parental cells neither accumulate ecmA transcripts nor form stalk cells. These findings indicate that constitutive PKA activity renders cells able to express prestalk-cell-specific genes under conditions where wild-type cells cannot do so, and hence that PKA is probably implicated in prestalk gene expression during normal development.

Zinc ions promote prestalk-to-stalk and prespore-to-stalk conversions inDictyostelium discoideum

To clarify the mechanism of stalk cell differentiation in Dictyostelium discoideum (strain NC4), we have examined the effects of Zn2+ on in vitro cell differentiation of prestalk and prespore cells isolated from normally formed slugs. Prestalk cells did not differentiate into stalk cells under submerged conditions, but in the presence of the stalk-inducing factor-1 (DIF-1) at 100 nM or Zn2+ at 5 mM, a small number of the cells (< 15%) differentiated into stalk cells. Interestingly, Zn2+ in combination with DIF-1 induced the prestalk-to-stalk conversion at high efficiencies (approx. 60%). Furthermore, isolated prespore cells were also converted to stalk cells at high efficiencies (approx. 50%) in the presence of both DIF-1 and Zn2+, while the conversion poorly occurred in the absence of Zn2+. These results indicate that Zn2+ may mimic some cellular interaction(s) which are required for stalk cell formation in this strain.

Zinc ions promote prestalk-to-stalk and prespore-to-stalk conversions in Dictyostelium discoideum

To clarify the mechanism of stalk cell differentiation in Dictyostelium discoideum (strain NC4), we have examined the effects of Zn 2+ on in vitro cell differentiation of prestalk and prespore cells isolated from normally formed slugs. Prestalk cells did not differentiate into stalk cells under submerged conditions, but in the presence of the stalk-inducing factor-1 (DIF-1) at 100 nM or Zn 2+ at 5 mM, a small number of the cells (< 15%) differentiated into stalk cells. Interestingly, Zn 2+ in combination with DIF-1 induced the prestalk-to-stalk conversion at high efficiencies (approx. 60%). Furthermore, isolated prespore cells were also converted to stalk cells at high efficiencies (approx. 50%) in the presence of both DIF-1 and Zn 2+, while the conversion poorly occurred in the absence of Zn 2+. These results indicate that Zn 2+ may mimic some cellular interaction(s) which are required for stalk cell formation in this strain.

Glycogen synthase kinase 3 regulates cell fate in dictyostelium

Extracellular cyclic AMP (cAMP) induces the formation of prespore cells in Dictyostelium but inhibits stalk cell formation. We have cloned gskA, which encodes the Dictyostelium homolog of glycogen synthase kinase 3 (GSK-3), and discovered that it is required for both cAMP effects. Disruption of gskA creates a mutant that aggregates but forms few spores and an abnormally high number of stalk cells. These stalk cells probably arise from an expanded prestalk B (pstB) cell population, which normally produces the basal disc of the fruiting body. In cultured mutant cells, cAMP neither inhibits pstB cell differentiation nor induces efficient prespore cell differentiation. We propose that cAMP acts through a common pathway that requires GSK-3 and determines the proportion of prespore and pstB cells.

Specific Induction by Zinc of Dictyostelium Stalk Cell Differentiation

Zinc is found in a variety of organisms and has been suggested to be essential for many cellular functions. We report here that zinc ions (Zn2+) have a specific and striking effect on cell differentiation in the simple eukaryote, Dictyostelium discoideum. In its asexual reproduction phase, solitary cells gather to form multicellular fruiting bodies consisting of spores and stalk cells. In vitro studies have revealed endogenous factors required for the differentiation of these two cell types. For stalk cell differentiation, these are cyclic AMP and a putative morphogen, a unique chlorinated alkylphenone named differentiation-inducing factor (DIF). With millimolar concentrations of Zn2+, however, cells neglect these requirements altogether and differentiate in vitro into mature stalk cells without the addition of cAMP and/or DIF, expressing stalk-specific markers, wst34 (protein) and ecmB (gene), but not ecmA. Zn2+-induced stalk cell formation is thus distinct from normal stalk cell formation and this system would provide a clue to elucidate the mechanism of stalk cell differentiation.

The effect of extracellular cyclic AMP on differentiation inducing factor (DIF)-dependant prestalk cell gene expression in monolayers of Dictyostelium is complex

Abstract An earlier finding that the ecmA and ecmB prestalk cell specific genes exhibited very different responses to cyclic AMP prompted the suggestion that cyclic AMP might act as the major spatial regulator of the prestalk cell developmental pattern in Dictyostelium. A more detailed kinetic analysis in monolayers of Dictyostelium has revealed that cyclic AMP inhibits the rate of expression of all three differentiation inducing factor (DIF) inducible genes, ecmA, ecmB and pDd26. After prolonged incubation, however, cyclic AMP enhances the levels of both ecmA and ecmB mRNAs, and nuclear run-on experiments suggest that cyclic AMP inhibits the degradation of both mRNA species. This complex response to cyclic AMP can explain the differential effects reported previously. Thus depending upon the experimental conditions, cyclic AMP can either enhance or reduce the apparent steady state level of a specific mRNA species. These results are not compatible with the earlier proposal that cyclic AMP is a spatial regulator of the prestalk developmental pattern. Although ecmA and ecmB accumulate rapidly in response to DIF, there is a lag in the accumulation of pDd26 mRNA and the induction requires protein synthesis. These results suggest that pDd26 transcription requires the accumulation of an additional factor(s). Inhibition of pDd26 mRNA accumulation by cyclic AMP also occurs during the lag period, suggesting the possibility that cyclic AMP inhibits the accumulation of the, as yet, unknown factor. The inhibitory effect of cyclic AMP on pDd26 gene expression is unaffected by caffeine, suggesting that inhibition does not involve adenylate cyclase activation. Finally the expression of pDd26 in the absence of cyclic AMP is unaffected by caffeine, suggesting that the inhibitory effect of cyclic AMP on stalk cell formation is not mediated at the level of pDd26 gene expression.

Prestalk cells in monolayer cultures exhibit two distinct modes of cellulose synthesis during stalk cell differentiation in Dictyostelium

ABSTRACT Stalk formation in Dictyostelium discoideum involves the synthesis of a stalk tube by the prestalk cell population and stalk cell walls by the individual prestalk cells. Cellulose is a major structural component of the stalk tube and stalk cell walls. The DIF-deficient strain HM44 was used to study the events of stalk formation in monolayer cultures. The induction of cellulose synthase activity was shown to require both DIF and cAMP. Microscopical observations of monolayer cultures using the cellulose-indicating fluorochrome Tinopal LPW demonstrated the presence in these cultures of two cellulose-containing materials: the stalk cell walls and an intercellular material found between cells and around cell clumps. The synthesis of intercellular material precedes that of stalk cell walls in induced cultures. Cells committed to stalk cell formation were delayed in doing so if they were switched to medium containing cAMP but no DIF. During this delay the cells synthesized large quantities of the intercellular material. The intercellular material was shown to be microfibrillar, was sensitive to cellulase, and labelled with a colloidal gold-conjugated cellulase. The intercellular material may have the same mode of cellulose synthesis as that involved in stalk tube formation. If so, that mode would be favored by DIF and cAMP in combination, whereas the cellulose synthesis involved in stalk cell wall formation would be DIF-dependent but delayed or repressed by cAMP.

Protein Phosphorylation during the Process of Prestalk-to-Stalk Conversion in Dictyostelium discoideum: (Dictyostelium discoideum/8-bromo cyclic AMP/stalk differentiation/protein kinase A/protein kinase inhibitor).

We have previously shown that 8-bromo cAMP (Br-cAMP) efficiently induces prestalk-to-stalk conversion in in vitro cultures of the cellular slime mold Dictyostelium discoideum, and that protein kinase A (PKA) plays an important role(s) in the conversion process (Kubohara et al. Exp. Cell Res. vol. 207, pp. 107-114, 1993). In the present study, we analyzed protein phosphorylation during the Br-cAMP-induced process, and identified two phosphoproteins, p60 and p40, that were specifically induced (phosphorylated) by Br-cAMP. The protein kinase inhibitors, staurosporine and K252a, which blocked the Br-cAMP-induced stalk cell formation, also inhibited the phosphorylations of these proteins. These results suggest that p60 and p40 may be the target proteins of PKA and thus be involved in the prestalk-to-stalk conversion.