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. EXPERIMENTAL APPROACH: P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca(2+) channel blocker. KEY RESULTS: Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3-3 µM) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca(2+) channel blocker; 100 nM) and a small interfering RNA for the T-type Ca(2+) channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO. CONCLUSIONS AND IMPLICATIONS: Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca(2+) channel gene.

Novel oligosaccharide has suppressive activity against human leukemia cell proliferation.

Various oligosaccharides containing galactose(s) and one glucosamine (or N-acetylglucosamine) residues with beta1-4, alpha1-6 and beta1-6 glycosidic bond were synthesized; Galbeta1-4GlcNH(2), Galalpha1-6GlcNH(2), Galalpha1-6GlcNAc, Galbeta1-6GlcNH(2), Galbeta1-4Galbeta1-4GlcNH(2) and Galbeta1-4Galbeta1-4GlcNAc. Galalpha1-6GlcNH(2) (MelNH(2)) and glucosamine (GlcNH(2)) had a suppressive effect on the proliferation of K562 cells, but none of the other saccharides tested containing GlcNAc showed this effect. On the other hand, the proliferation of the human normal umbilical cord fibroblast was suppressed by none of the saccharides other than GlcNH(2). Adding Galalpha1-6GlcNH(2) or glucosamine to the culture of K562 cell, the cell number decreased strikingly after 72 h. Staining the remaining cells with Cellstain Hoechst 33258, chromatin aggregation was found in many cells, indicating the occurrence of cell death. Furthermore, all of the cells were stained with Galalpha1-6GlcNH-FITC (MelNH-FITC). Neither the control cells nor the cells incubated with glucosamine were stained. On the other hand, when GlcNH-FITC was also added to cell cultures, some of them incubated with Galalpha1-6GlcNH(2) were stained. The difference in the stainability of the K562 cells by Galalpha1-6GlcNH-FITC and GlcNH-FITC suggests that the intake of Galalpha1-6GlcNH(2) and the cell death induced by this saccharide is not same as those of glucosamine. The isolation of the Galalpha1-6GlcNH(2) binding protein was performed by affinity chromatography (melibiose-agarose) and LC-MS/MS, and we identified the human heterogeneous ribonucleoprotein (hnRNP) A1 (34.3 kDa) isoform protein (30.8 kDa). The hnRNP A1 protein was also detected from the eluate(s) of the MelNH-agarose column by the immunological method (anti-hnRNP-A1 and HRP-labeled anti-mouse IgG (gamma) antibodies).

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.

N-methyl-D-aspartate receptor-dependent and -independent cytotoxic effects of Dictyostelium discoideum differentiation-inducing factor-1 on rat cortical neurons.

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (small lipophilic hormone) that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. Recent studies have revealed that DIF-1 inhibits growth and induces the differentiation of mammalian tumor cells. The present study examines the effects of DIF-1 on rat cortical neurons in primary culture. We found that DIF-1 induced rapid neuronal cell death. The release of lactate dehydrogenase (LDH), as an indicator of cell death, increased dose-dependently with DIF-1. The release of LDH was inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonists MK801 and AP5, suggesting that the NMDA receptor is involved in the induction of cell death by DIF-1. However, glutamate cytotoxicity could not explain the entire action of DIF-1 on neurons because the estimated concentration of glutamate around DIF-1-treated neurons was below 50 microM and DIF-1 caused more severe cell death than 500 microM glutamate. We discovered that another portion of DIF-1 cytotoxicity is independent of the NMDA receptor; that is, coaddition of DIF-1 and MK801 induced dendritic beading and increased expression of the immediate early genes c-fos and zif/268. These results indicate that DIF-1 induces rapid cell death via both NMDA receptor-dependent and -independent pathways in rat cortical neurons.

Caspase-independent apoptosis induced by differentiation-inducing factor of Dicytostelium discoideum in INS-1 cells.

Differentiation-inducing factor (DIF) is a lipophilic hormone of Dicytostelium discoideum and has been shown to exert diverse effects in mammalian cells. We investigated the effect of DIF on cell viability in insulin-secreting INS-1 cells. DIF induced cell death in a dose-dependent manner. In DIF-treated cells, nuclear condensation and shrinkage of the cell body were observed. After 6 h of DIF treatment, cells became Tdt-mediated dUTP-biotin nick end-labeling-positive, and DNA ladder formation was detected, indicating that DIF induced apoptosis in these cells. DIF did not activate caspase-3, a key enzyme mediating apoptotic signals generated by various agents. Furthermore, DIF-induced cell death was not affected by Z-asp-2, 6-dichlorobenzoyloxymethylketone, a broad inhibitor of the caspases. As is the case in other types of cells, DIF increased cytoplasmic free calcium concentration in INS-1 cells. However, DIF-induced cell death was not affected by chelating intracellular free calcium by 1, 2-bis(2-aminoophenoxy)ethane-N, N, N, N-tetra acetic acid (BAPTA). These results indicate that DIF induces apoptosis in INS-1 cells by a mechanism independent of caspase-3. DIF-induced elevation of cytoplasmic calcium does not mediate the effect of DIF on cell death.

A novel role of differentiation-inducing factor-1 in Dictyostelium development, assessed by the restoration of a developmental defect in a mutant lacking mitogen-activated protein kinase ERK2.

It has been previously reported that the differentiating wild-type cells of Dictyostelium discoideum secrete a diffusible factor or factors that are able to rescue the developmental defect in the mutant lacking extracellular signal-regulated kinase 2 (ERK2), encoded by the gene erkB. In the present study, it is demonstrated that differentiation-inducing factor-1 (DIF-1) for stalk cells can mimic the role of the factor(s) and the mechanism of the action of DIF-1 in the erkB null mutant is also discussed. The mutant usually never forms multicellular aggregates, because of its defect in cyclic adenosine monophosphate (cAMP) signaling. In the presence of 100 nM DIF-1, however, the mutant cells formed tiny slugs, which eventually developed into small fruiting bodies. In contrast, DIF-1 never rescued the developmental arrest of other Dictyostelium mutants lacking adenylyl cyclase A (ACA), cAMP receptors cAR1 and cAR3, heterotrimeric G-protein, the cytosolic regulator of ACA, or the catalytic subunit of cAMP-dependent protein kinase (PKA-C). Most importantly, it was found that DIF-1 did not affect the cellular cAMP level, but rather elevated the transcriptional level of pka during the development of erkB null cells. These results suggest that DIF-1 may rescue the developmental defect in erkB null cells via the increase in PKA activity, thus giving the first conclusive evidence that DIF-1 plays a crucial role in the early events of Dictyostelium development as well as in prestalk and stalk cell induction.

Differentiation-inducing factor-1, a morphogen of dictyostelium, induces G(1) arrest and differentiation of vascular smooth muscle cells.

Differentiation-inducing factor-1 (DIF-1) is a morphogen that induces differentiation of DICTYOSTELIUM: Recently, DIF-1 has been shown to inhibit proliferation and induce differentiation in tumor cells, although the underlying mechanisms remain unknown. In this study, we examined the effects of DIF-1 on the proliferation and differentiation of vascular smooth muscle cells, to explore novel therapeutic strategies for atherosclerosis. DIF-1 nearly completely inhibited DNA synthesis and cell division in mitogen-stimulated cells. DIF-1 inhibited the phosphorylation of the retinoblastoma protein and the activities of cyclin-dependent kinase (Cdk) 4, Cdk6, and Cdk2, which phosphorylate the retinoblastoma protein. DIF-1 strongly suppressed the expression of cyclins D1, D2, and D3, as well as those of cyclins E and A, which normally began after that of the D-type cyclins. The mRNAs for the smooth muscle myosin heavy chains SM1 and SM2 were expressed in quiescent cells in primary culture, and these expression levels decreased after mitogenic stimulation. In the presence of DIF-1, the rate of the reduction was significantly decelerated. Moreover, the addition of DIF-1 to dedifferentiated cells induced the expressions of SM1 and SM2, accompanied by a reduction in the level of SMemb, a nonmuscle-type myosin heavy chain. Therefore, DIF-1 seemed to interrupt a very early stage of G(1) probably by suppressing the expressions of the D-type cyclins. Furthermore, this compound may prevent phenotypic modulation and induce differentiation of vascular smooth muscle cells.

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 ([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.

The putative morphogen, DIF-1, of Dictyostelium discoideum activates Akt/PKB in human leukemia K562 cells.

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.

Cotylenin A, a plant-growth regulator, induces the differentiation in murine and human myeloid leukemia cells.

Protein factors playing a significant part in differentiation and development have been recently elucidated. However, low molecular factors which also seem to be essential remain still unknown, although only retinoic acid has become such a candidate. Cotylenins had been isolated as the plant-growth regulators, and have been found to affect a number of physiological processes of higher plants. Here we report that at the concentrations above 12.5 microg/ml (20 microM) cotylenin A induced the functional and morphological differentiation in murine (M1) and human myeloid leukemia (HL-60) cells. Although cotylenin A has some similarity to PMA both in carbotricyclic diterpene structure and in biological activity (i.e. differentiation-induction of HL-60 cells into macrophages), the activation of PKC and the elevation of Ca2+-levels by cotylenin A were not observed. Quite recently it has been reported that fusicoccin (closely related to cotylenin A)-targets are 14-3-3 proteins, which are at the crosspoint of a huge array of signalling and regulatory pathways. These results suggest that cotylenin A might become a useful tool for the elucidation of molecular mechanisms of differentiation and development.

DIF-1, putative morphogen of D. 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 mold Dictyostelium discoideum. In this study, we have examined the effects of DIF-1 on the human leukemia HL-60 cells. DIF-1 at 10-40 microM suppressed cell growth in a dose-dependent manner, and approximately 50% growth inhibition was attained with 15-20 microM 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 microM DIF-1. These concentrations of DIF-1 also raised [Ca2+]i in 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 microM) 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.

Bradykinin increases intracellular free Ca2+ concentration and promotes insulin secretion in the clonal beta-cell line, HIT-T15.

We have examined the effects of bradykinin (BK) on both the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in the hamster beta-cell line, HIT-T15 cells. BK evoked a rise in [Ca2+]i in a dose-dependent manner. This response was suppressed by neomycin, suggesting that BK mobilizes Ca2+ from intracellular store via promotion of the phosphatidyl inositol turnover. Furthermore, BK also evoked insulin secretion. Both the BK-evoked rise in [Ca2+]i and insulin secretion were suppressed by the BK2 receptor antagonist, but not by the BK1 receptor antagonist. These results indicate that BK increases [Ca2+]i via BK2 receptor, thereby promoting insulin secretion in HIT-T15 cells.

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 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.

DIF-1, morphogen of Dictyostelium discoideum, induces the erythroid differentiation in murine and human leukemia cells.

The structure of morphogen of Dictyostelium discoideum, DIF-1, which was elucidated by Morris et al. proved to be closely similar to that of differanisole A which had been isolated by us from the metabolites of a simple eucaryote, Chaetomium, as the differentiation-inducer of murine and human undifferentiated tumor cells. This fact seemed to suggest the molecular structure playing an important part in differentiation and development beyond species. We have already examined whether differanisole A induces the differentiation in Dictyostelium discoideum, and it was morphologically and biochemically confirmed. In this study, we examined whether DIF-1 induces the differentiation in murine and human leukemia cells. Above the concentration of 2 micrograms/ml (6.5 microM), DIF-1 induced the differentiation in murine erythroleukemia B8 cells and in human leukemia K562 cells into the hemoglobin-synthesizing erythrocyte-like cells.

Differentiation-inducing factor of D. discoideum raises intracellular calcium concentration and suppresses cell growth in rat pancreatic AR42J cells.

DIF (differentiation-inducing factor) is a putative morphogen that induces stalk cell differentiation in the lower eukaryote, Dictyostelium discoideum. In this study, we have examined the effects of DIF on growth and the intracellular calcium concentration ([Ca2+]i) in rat pancreatic acinar AR42J cells. Growth of AR42J cells was inhibited when DIF was present in the media, and approximately 50% growth inhibition was attained with 20 microM DIF. DIF was also found to raise [Ca2+]i in a dose-dependent manner (1-40 microM), both in the presence and absence of extracellular Ca2+. These results suggest that DIF elicits both calcium influx from the extracellular space and calcium release from intracellular pool(s), thereby inhibiting cell growth in AR42J.

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.

Cytoplasmic Ca2+ and H+ concentrations determine cell fate in Dictyostelium discoideum.

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.

Analysis of the maturation process of prestalk cells in Dictyostelium discoideum.

The requirements for the terminal differentiation process of the stalk pathway (from prestalk to stalk) of Dictyostelium discoideum were analyzed by using a low-cell-density culture system with prestalk cells isolated from normally developed slugs. Any of the substances, such as differentiation-inducing factor-1 (DIF-1), DIF-2, dimethyloxazolidinedione, and adenosine, that had been shown to promote prestalk/stalk differentiation did not cause an efficient and consistent induction of prestalk-to-stalk conversion, either alone or in combination. However, the addition of 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP) resulted in high efficiency (80-90%) of stalk cell formation which accompanied the accumulation of a stalk-specific protein, wst34. The maturation process was inhibited by Ca2+ but not by Mg2+. More importantly, the Br-cAMP-induced stalk cell differentiation was neither inhibited nor promoted by DIF-1, cAMP, or ammonia and occurred even at very low cell densities if only Br-cAMP was supplied. Since Br-cAMP is though to penetrate into the cells and to activate the intracellular protein kinase A, the present results suggest that the activation of protein kinase A is sufficient for prestalk-to-stalk conversion.

Differanisole A, an inducer of the differentiation of Friend leukemic cells, induces stalk cell differentiation in Dictyostelium discoideum.

Differanisole A isolated from the conditioned medium of a soil microorganism, Chaetomium strain RB-001, is an inducer of the differentiation of the Friend leukemic cells (mouse leukemia cells). The chemical structure of this substance is very similar to that of stalk cell differentiation-inducing factor (DIF) isolated from the cellular slime mould, Dictyostelium discoideum. We examined the effects of differanisole A on Dictyostelium HM44 cells, a mutant strain which is defective in DIF production, and found this substance to be an inducer of stalk cell differentiation in D. discoideum.