Hinokitiol induces differentiation of teratocarcinoma F9 cells

Hinokitiol, a constituent of the wood of Chamaecyparis taiwanensis, was found to induce differentiation of teratocarcinoma F9 cells. When examined by the agar-overlay method, in which expression of plasminogen activator as a differentiation marker protein was detected, this compound exhibited a dose- and time-dependent induction. Induction of differentiation by hinokitiol occurred irreversibly and required its addition for more than 12h. Among its structure-related compounds tested, tropolone and two colchicine-related compounds exerted potent activities comparable to that of hinokitiol. These findings indicate that free tropolone structure in the molecules plays an essential role in inducing differentiation of F9 cells. Hinokitiol showed a strong inhibitory effect of DNA synthesis in very early stages of culture, suggesting that this effect may be responsible for triggering differentiation of F9 cells.     

Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation

Single endothelial cells (EC) seeded in suspension culture rapidly undergo apoptosis. Addition of survival factors, such as VEGF and FGF-2, does not prevent apoptosis of suspended EC. However, when cells are allowed to establish cell-cell contacts, they become responsive to the activities of survival factors. These observations have led to the development of a three-dimensional spheroid model of EC differentiation. EC spheroids remodel over time to establish a differentiated surface layer of EC and a center of unorganized EC that subsequently undergo apoptosis. Surface EC become quiescent, establish firm cell-cell contacts, and can be induced to express differentiation antigens (e.g., induction of CD34 expression by VEGF). In contrast, the unorganized center spheroid cells undergo apoptosis if they are not rescued by survival factors. The responsiveness to the survival factor activities of VEGF and FGF-2 was not dependent on cell shape changes since it was retained after cytochalasin D treatment. Taken together, these findings characterize survival factor requirements of unorganized EC and indicate that polarized surface EC differentiate to become independent of exogenous survival factors. Furthermore, they demonstrate that spheroid cell culture systems are useful not just for the study of tumor cells and embryonic stem cells but also for the analysis of differentiated functions of nontransformed cells.     

Protein phosphorylation required for the formation of E2F complexes regulates N-myc transcription during differentiation of human embryonal carcinoma cells

The human embryonal carcinoma (EC) cell line NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). The N-myc gene is expressed at a high level in the undifferentiated cells, but the level decreased steeply after 12-24 h HMBA treatment, returning to its original level after 48 h. The alteration in the N-myc level was well correlated with the formation of complexes with the E2F motif in the N-myc promoter region, and no complex was formed with cell extracts prepared from cells treated with HMBA for 12-24 h. The absence of E2F complexes during this period was caused by an inhibitor generated by a phosphatase reaction. Treatment of the 12-h extract with a cyclic AMP-dependent protein kinase resulted in the formation of E2F complexes, and treatment of the undifferentiated (0 h) and 48-h extracts with a calf intestinal phosphatase abolished complex formation completely. An inhibitor generated by the 0-h extract after treatment with a phosphatase inhibited E2F complex formation by the untreated 0-h extract in the presence of phosphatase inhibitors, okadaic acid and sodium vanadate. One of the two E2F complexes in the undifferentiated cells contained cyclin A, but the complex with similar mobility, formed after the transient decrease in the N-myc level, did not.     

The Na+/H+ antiporter potentiates growth and retinoic acid-induced differentiation of P19 embryonal carcinoma cells

The Na+/H+ exchanger is a ubiquitous plasma membrane protein that is responsible for pH regulation and is activated by growth factors. We examined the role of the Na+/H+ exchanger in cell growth and differentiation. Treatment of P19 cells with the Na+/H+ exchanger inhibitor Hoe 694 eliminated retinoic acid-induced differentiation in this cell line. We developed a P19 embryonal carcinoma cell line that was deficient in the Na+/H+ antiporter. Na+/H+ exchanger-deficient cells were reduced in the rate of cell growth and this effect was enhanced by the removal of added HCO3- and by reducing extracellular pH. The antiporter-deficient cells were also markedly deficient in their ability to differentiate to neuronal-like cells and recovered this ability when the Na+/H+ antiporter was reintroduced. The results show that the absence of Na+/H+ antiport as a pH regulatory mechanism can result in deficiencies in both cell growth and differentiation in embryonal carcinoma cells.     

In vitro induction of differentiation by ginsenoides in F9 teratocarcinoma cells

The aim of this study was to determine the ability of the ginsenosides, extracts of Panax ginseng C.A. Meyer, to cause differentiation of F9 teratocarcinoma stem cells as a model system. F9 stem cells cultured in the presence of the ginsenosides together with dibutyryl cyclic AMP (dbcAMP) became parietal endoderm-like cells. Moreover, the expression of differentiation marker genes, such as laminin B1 and type IV collagen, was increased after treatment with the ginsenosides. Among the various purified ginsenosides, Rh1 and Rh2 were the most effective at causing differentiation of F9 cells. Since ginsenosides and glucocorticoid hormone have similar chemical structures, we examined the possibility of the involvement of a glucocorticoid receptor (GR) in the differentiation process induced by the ginsenosides. According to Southwestern blot analysis, a 94 kDa protein regarded as a GR was detected in F9 cells cultured in the medium containing the ginsenosides Rh1 or Rh2. In addition, F9 stem cells treated with the ginsenosides Rh1 or Rh2 and with RU486, a glucocorticoid antagonist with a high affinity for the GR, did not differentiate into endoderm cells morphologically, and the expression of laminin B1 gene was not induced in these cells. In a gel mobility shift assay, protein factors capable of binding to the glucocorticoid responsive element (GRE) specifically were detected in nuclear extracts of the ginsenoside-treated F9 cells. Moreover, overexpression of GR by cotransfection of GR expression vector and GRE-luciferase vector enhanced the transactivation activity of GRE promoter in the presence of ginsenosides Rh1 or Rh2 and was further augmented by dbcAMP. In addition, ginsenosides Rh1 and Rh2 bound to a GR assessed by whole-cell binding assay, even though the specific binding affinity was weaker compared to dexamethasone. Based on these data, we suggest that the ginsenosides Rh1 and Rh2 cause the differentiation of F9 cells and the effects of ginsenosides might be exerted via binding with a GR or its analogous nuclear receptor.     

IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages

Human monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-13 for 7 days differentiate into cells with the morphology and function of dendritic cells (DC). We have investigated the effect of IL-10 on this differentiation pathway. In the presence of IL-10 cells did not develop DC morphology, did not express CD1a and had lower levels of MHC class II. IL-10 promoted the differentiation of large cells with the morphology, cytochemistry and membrane phenotype of macrophages, including staining for nonspecific esterase and high levels of CD14, CD16 and CD68. The effect of IL-10 was dose dependent and was best appreciated when the cytokine was added at the initiation of the culture, as addition on day 3 was less inhibitory. When added to already differentiated DC on day 6, IL-10 caused only a modest reduction of MHC class II and CD1a expression, and no acquisition of the macrophage markers CD14, CD16 and CD68. Prolonged incubation up to 5 days with IL-10 did not induce a shift of differentiated DC to macrophages. On the other hand, the macrophages obtained by culturing for 7 days with GM-CSF+IL-13+IL-10 did not shift to DC upon removal of IL-10 for up to 3 days. Thus, the effect of IL-10 on monocyte differentiation, occurs only at the precursor level and confers an irreversible phenotype. From a functional point of view, cells cultured in the presence of IL-10 were poor stimulators of allogeneic cord blood T cells in mixed lymphocyte reaction (MLR) and presented tetanus toxin (TT) to specific T cell lines with much less efficiency than control DC. In contrast, IL-10-cultured DC showed 7 times greater endocytosis of FITC-dextran. This increased endocytosis was mostly mediated via the mannose receptor, as demonstrated by blocking with unlabeled mannose. In conclusion, IL-10 inhibits DC differentiation from monocytes and, in a substantial proportion of the cells, promotes the differentiation to mature macrophages. Intriguingly, IL-10 inhibits antigen presentation while it stimulates endocytic activity.     

Wortmannin enhances CPP32-like activity during neuronal differentiation of P19 embryonal carcinoma cells induced by retinoic acid

P19 EC cells undergoes apoptosis during neuronal differentiation induced by retinoic acid. Two CPP32-like proteases, CPP32 and Mch-3, are expressed in untreated and retinoic acid-treated P19 EC cells. CPP32-like activity is remarkably increased in apoptosis during neuronal differentiation of P19 EC cells. Inhibition of CPP32-like proteases prevents apoptosis, suggesting that activation of CPP32-like proteases play central roles in the apoptosis during neuronal differentiation of P19 EC cells. Wortmannin, PI-3K inhibitor, enhances the CPP32-like activity of the retinoic acid-treated P19 EC cells. PI-3K may be involved in the apoptosis during neuronal differentiation as negative regulator.     

Galpha12 and Galpha13 mediate differentiation of P19 mouse embryonal carcinoma cells in response to retinoic acid

P19 mouse embryonal carcinoma cells can be stimulated to differentiate into endodermal-like, mesodermal-like, and neuronal-like phenotypes in response to specific morphogens. At low concentrations, retinoic acid stimulates P19 embryonal cells to differentiate to cells displaying an endodermal phenotype, whereas at higher concentrations it stimulates differentiation to neuroectoderm. The Galpha12 and Galpha13 subunits of heterotrimeric G-proteins are expressed in the embryonal P19 cells and stimulated in response to retinoic acid as the cells differentiate to endodermal or neuroectodermal phenotypes. Suppression of the expression of either Galpha12 or Galpha13 by antisense RNA is shown to promote cell detachment from substratum and apoptosis. Expression of the constitutively active, mutant form of Galpha12 (Q229L), in contrast, stimulates loss of the embryonal phenotype. Expression of the constitutively active form of Galpha13 (Q226L) stimulates differentiation of the cells from embryonal to endodermal, in the absence of retinoic acid. Thus, both Galpha12 and Galpha13 are essential to stimulation of cell differentiation by retinoic acid. Deficiency of either Galpha12 or Galpha13 increases programmed cell death.     

The distal cytoplasmic domain of the erythropoietin receptor induces granulocytic differentiation in 32D cells

The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood 89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)-dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation.     

T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation

The central role of type-2 helper T (Th2) cells in the development of allergic responses and immune responses against helminthic parasites is well documented. The differentiation of Th2 cells from naive T cells requires both the recognition of antigen by T cell antigen receptors (TCR) and the activation of downstream signal-transduction molecules of the interleukin 4 receptor (IL-4R) pathway, including Jak1, Jak3, and STAT6. Little is known, however, about how these two distinct pathways cooperate with each other to induce Th2 cells. Here, we use a T cell-specific H-Ras-dominant-negative transgenic mouse to show that TCR-mediated activation of the Ras/mitogen-activated protein kinase pathway alters IL-4R function and is required for Th2 cell differentiation. The enhancement of IL-4R signaling seems to be a consequence of both direct "crosstalk" with the TCR signaling pathway and increased protein expression of downstream signaling molecules of the IL-4R pathway. Therefore, successful Th2 differentiation depends on the effectiveness of the TCR-mediated activation of the Ras/mitogen-activated protein kinase pathway in modifying the IL-4R-mediated signaling pathway.