Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia cells (Ml) were induced to differentiate into mature macrophages and granulocytes by various inducers. The differentiated Ml cells synthesized and released prostaglandins, whereas untreated Ml cells did not. When the cells were prelabelled with [¹⁴C]arachidonate, the major prostaglandins released into the culture media were found to be prostaglandin E₂, D₂, and F_2α in an early stage of differentiation, but the mature cells produced predominantly prostaglandin E₂. The synthesis and release of prostaglandins were completely inhibited by indomethacin. Dexamethasone, a potent inducer of differentiation of Ml cells, did not induce production of prostaglandins in resistant Ml cells that could not differentiate even with a high concentration of dexamethasone. These results suggest that production of prostaglandins in Ml cells is closely associated with differentiation of the cells. Homogenates of dexamethasone-treated Ml cells converted arachidonate to prostaglandins, but this conversion was scarcely observed with homogenates of untreated Ml cells. Dexamethasone and the other inducers stimulated the release of arachidonate from phospholipids. Therefore, induction of prostaglandin synthesis during differentiation of Ml cells may result from induction of prostaglandin synthesis activity and stimulation of the release of arachidonate from cellular lipids. Lysozyme activity, which is a typical biochemical marker of macrophages, was induced in Ml cells by prostaglandin E₂ or D₂ alone, as well as by inducers of differentiation of the cells, but it was not induced by arachidonate or prostaglandin F_2α. These results suggest that prostaglandin synthesis is important in differentiation of myeloid leukemia cells.     

Induction of lysozyme activity by adenosine 3':5' cyclic monophosphate in cultured mouse myeloid leukemic cells.

Mouse myeloid leukemic cells(Ml) could be induced by glucocorticoids to form Fc receptors, phagocytize, migrate in agar, induce lysosomal enzyme activities, and change into forms that were morphologically similar to macrophages and granulocytes. Adenosine 3′:5′ cyclic monophosphate also induced lysosomal enzyme activities, but not the other differentiation-associated properties. The induction of lysozyme activity was marked, the activity reaching about 400 times the initial activity at 5 days after treatment. This suggests that adenosine 3′:5′ cyclic monophosphate may be important in induction of lysozyme activity during differentiation of the cells.     

Purification of host DNA synthesis-suppressing factor (DSF) produced by infection with measles virus.

Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.     

Purification of Host DNA Synthesis-Suppressing Factor (DSF) Produced by Infection with Measles Virus

Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.     

Differentiation of a cell line of mouse myeloid leukemia : I. Simultaneous induction of lysosomal enzyme activities and phagocytosis

Induction of phagocytic activity in the Ml cell line of mouse myeloid leukemia, on being exposed to a conditioned medium from cultured embryo cells, was accompanied by an increment in the activities of both lysosomal acid phosphatase and acid protease. The activity of these lysosomal enzymes, as well as that of phagocytosis, was not induced when Ml cells were incubated either with the conditioned medium subjected to heat treatment or in the presence of 5-bromodeoxyuridine (BUdR). The levels of these induced enzyme activities in Ml cells were comparable to those in normal mouse peritoneal macrophages. The lysosomal enzyme activity in Mm-1 cells, which were spontaneously differentiated from Ml cells and exhibiting a higher phagocytic activity, were reminiscent of those in peritoneal macrophages. Based on these observations, it was concluded that both phagocytosis and lysosomal enzyme activity occur simultaneously during the course of differentiation. This differentiation, morphological or functional, in Ml cells in the presence of the conditioned medium was further supported by biochemical evidence.     

Characterization of a factor inducing differentiation of mouse myeloid leukemic cells purified from conditioned medium of mouse Ehrlich ascites tumor cells

A factor inducing differentiation of mouse myeloid leukemic cells (MI) into macrophages was purified to apparent homogeneity from 168 1 of CM of Ehrlich ascites tumor cells. The purified factor was half-maximally active at 2 X 10(-11) M. The factor was analyzed by radioiodination, SDS-polyacrylamide gel electrophoresis and autoradiography. Its Mr was 40 000-50 000. On reduction, the factor lost activity, but showed no subunit structure. Treatment of the factor with endo-beta-N-acetylglucosaminidase F, but not endo-beta-N-acetylglucosaminidase H, gave rise to a molecule of Mr 20 000-28 000. The activity of the factor from Ehrlich cells was completely neutralized by antiserum to the factor of Mr 50 000-70 000 from mouse fibroblast L929 cells.     

The role of interferon-gamma in induction of differentiation of human myeloid leukemia cell lines, ML-1 and HL-60

Highly purified natural interferon-gamma (IFN-gamma) induced differentiation having characteristics that are associated with the human promyelocytic leukemia cell line, HL-60. Monoclonal antibody to INF-gamma neutralized its activity. However, the natural IFN-gamma had almost no inducing activity in ML-1, a human myeloblastic leukemia cell line. Similar results were obtained using recombinant IFN-gamma. Mitogen stimulated human leukocyte conditioned medium (LCM) induced differentiation of both ML-1 and HL-60 cells. After treatment of LCM with monoclonal antibody to IFN-gamma, LCM activity was reduced more than 50% in ML-1 cells, and 80% in HL-60 cells. Even if IFN-gamma was eliminated from LCM by affinity chromatography, the LCM induced differentiation of ML-1 and HL-60 cells, but IFN-gamma markedly enhanced the ML-1 cell differentiation induced by IFN-gamma free LCM. The results suggest that leukocytes produce differentiation inducing factor(s) other than IFN-gamma, and that IFN-gamma is both an inducer and an enhancer of induction of human myelogenous leukemia cells.     

Murine pregnancy decidua produces a unique immunosuppressive molecule related to transforming growth factor beta-2

Non-T small lymphocytic suppressor cells in murine allopregnancy release a potent immunosuppressive factor in vitro that is neutralized by rabbit anti-transforming growth factor (TGF)-beta. Previous studies have suggested that the decidual suppressor factor (DSF) is smaller than TGF-beta 1, and in this paper, we show that DSF on HPLC-sieving columns also elutes later than TGF-beta 2. Nevertheless, DSF has the ability to promote anchorage-independent growth of NRK fibroblasts similar to TGF-beta s. Using turkey antibodies specific for TGF-beta 1 or beta 2, we show that DSF is related to TGF-beta 2 rather than TGF-beta 1, and this relationship was confirmed by using a panel of murine mAb to TGF-subtypes. PAGE and Western blotting showed that the TGF-beta 2-reactive molecules in HPLC-purified DSF was slightly smaller than TGF-beta 2 and approximately 20 to 23 kDa. The DSF molecule is therefore closely related to TGF-beta 2 but as released from decidua, differs in size. The TGF-beta 2-related decidual suppressor factor was also obtained from the decidua of synpregnant C.B.-17 severe combined immune deficiency (SCID) and pregnant SCID-BG (C57BL/6 background) mice, confirming the lack of T or B cell dependence of DSF production and the generality of production of a TGF-beta-related suppressor factor by decidua associated with successful pregnancy in mice.     

Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells

Experiments were conducted to purify the differentiation-inducing factor (D-factor), which induces differentiation of mouse myeloid leukemic cell line, Ml, into macrophage-like cells, in a conditioned medium of guinea pig peritoneal macrophages stimulated with lipopolysaccharide. On gel filtration under high performance liquid column chromatography (HPLC), D-factor eluted at the position of 45-15 KD. By the subsequent separation on DEAE HPLC the D-factor activity disappeared. However, in the presence of recombinant human IL 1 alpha the D-factor activity appeared at a position where tumor necrosis factor (TNF) eluted. Even after fractionation on hydroxyapatite HPLC the IL 1-dependent D-factor was co-chromatographed with TNF. Recombinant human TNF as well as the partially purified guinea pig TNF induced differentiation of Ml cells in conjunction with either the partially purified guinea pig IL 1 or recombinant human IL 1 alpha, although these factors by themselves did not induce differentiation. These findings suggest that a part of D-factor activity in the conditioned medium resulted from the cooperative effects between TNF and IL 1.     

Differentiation and production of colony-stimulating factor induced by immunostimulants in a leukemia cell line

Immunostimulants from various microorganisms were tested on a myeloid leukemia cell line (Ml) for the ability to induce production of CSF and to cause differentiation of these cells. Based on their activities, the compounds were divided into two general classes: those inducing extensive cellular differentiation and those devoid of this effect. The stimulants which were active in this regard always produced large quantities of CSF, whereas those devoid of a differentiating effect did not cause CSF production. Even the potent stimulants had no effect on the D^? subline, in which the differentiation was not inducible.     

Paradoxical stimulation of both lipocortin and prostaglandin production in human amnion cells by dexamethasone

Glucocorticoids inhibit prostaglandin biosynthesis by inducing the formation of lipocortins. In human amnion cells dexamethasone elicited a concentration-dependent increase in prostaglandin production and raised intracellular lipocortin 1 concentrations. Dexamethasone could also potentiate the epidermal growth factor (EGF)-induced stimulation of prostaglandin production. EGF alone or in combination with dexamethasone increased lipocortin 1 formation in amnion cells. Human amnion cells may provide a unique insight into interactions between glucocorticoids, lipocortin and eicosanoid biosynthesis.     

Anti-proliferative activity of disulfiram through regulation of the AKT-FOXO axis: A proteomic study of molecular targets

Due to its potent anti-tumor activity, well-investigated pharmacokinetic properties and safety profile, disulfiram (DSF) has emerged as a promising candidate for drug repurposing in cancer therapy. Although several molecular mechanisms have been proposed for its anti-cancer effects, the precise underlying mechanisms remain unclear. In the present study, we showed that DSF inhibited proliferation of cancer cells by inducing reactive oxygen species (ROS) production, a G1 cell cycle arrest and autophagy. Moreover, DSF triggered apoptosis via suppression of the anti-apoptotic protein survivin.To elucidate the mechanisms for the anti-proliferative activities of DSF, we applied a 2-DE combined with MALDI-TOF-MS/MS analysis to identify differentially expressed proteins in breast cancer cells upon treatment with DSF. Nine differentially expressed proteins were identified among which, three candidates including calmodulin (CaM), peroxiredoxin 1 (PRDX1) and collagen type I alpha 1 (COL1A1) are involved in the regulation of the AKT signaling pathway. The results of western blot analysis confirmed that DSF inhibited p-AKT, suggesting that DSF induces its anti-tumor effects via AKT blockade. Moreover, we found that DSF increased the mRNA levels of FOXO1, FOXO3 and FOXO4, and upregulated the expression of their target genes involved in G1 cell cycle arrest, apoptosis and autophagy. Finally, DSF potentiated the anti-proliferative effects of well-known chemotherapeutic agents such as arsenic trioxide (ATO), doxorubicin, paclitaxel and cisplatin. Altogether, these findings provide mechanistic insights into the anti-growth activities of DSF.     

Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae

Summary A chemostat culture system was investigated in order to produce protease by Aspergillus species effectively in the presence of 10% NaCl which was added to avid bacterial contamination. A salt tolerant fungus Aspegillus oryzae NISL 1913 produced protease even in the presence of 10% NaCl. The protease production by this strain was accelerated by proteins. Isolated soy protein or defatted soybean fluor (DSF) was used as a nitrogen source and an inducer of protease production, and starch was used as a carbon source. Continuous protease production was performed in a carbon-limited chemostat culture (dilution rate = 0.02). The maximum activity reached 2200 protease units (PU)/ml of the culture broth (130 PU/mg dry weight) with DSF as a nitrogen source. The culture could be continued for more than 50 days without any bacterial contamination.     

T cells produce TRF in response to Con A and factors in T cell hybridoma supernatants

In recent experiments, a soluble factor (TRF) that mediates the differentiation of anti-immunoglobulin (Ig)-activated B cells to Ig-secreting cells has been identified. TRF works in concert with a growth factor, probably IL 2, in the induction of activated B cells. In previous studies, TRF was identified in culture supernatants of activated T cells and accessory cells, and thus the cellular source (T cell or accessory cell) of the factor was not determined. In the present studies, we succeeded in inducing the production of TRF by T cell populations from which accessory cells had been vigorously depleted. Lymph node cells were depleted of accessory cells by nylon wool adherence and anti-Ia and complement treatment; these cells were activated with Con A and a T cell hybridoma supernatant that contains IL 2. Supernatants from these activated T cell cultures supported the differentiation of anti-Ig-activated B cells to Ig secreting cells. These results show that T cells produce the differentiation factor, and further that they do so in response to ligand (Con A) plus a T cell-derived factor.     

Three different signals are required for the induction of cytolytic T lymphocytes from resting precursors

The requirement for the signals in induction of cytolytic T lymphocytes (CTL) has been investigated. C57BL/6 X CBA/T6 F1 spleen cells stimulated with the lectin leukoagglutinin (L-A) failed to show CTL activity in a PHA-facilitated assay, although L-A-activated splenic T cells were able to respond to T cell growth factor (TCGF). Concanavalin A (Con A) on the other hand was able to induce cytolytic activity from CTL-P, as well as to render splenic T cells responsive to TCGF. Furthermore, L-A-activated splenic T cells could generate cytolytic activity upon subsequent culture in secondary mixed leukocyte culture supernatant (2 degrees MLC SN). In contrast, EL-4-derived SN (EL-4 SN) was unable to induce cytolytic activity from L-A-activated spleen cells. In addition, proliferation of L-A-activated spleen cells cultured in EL-4 SN was similar to those cultured in 2 degrees MLC SN. Nonactivated spleen cells were totally unresponsive to both SN in proliferation and generation of CTL. Analysis of T cell clones for the production of a factor necessary for induction of cytolytic activity revealed that both cytolytic and noncytolytic T cell clones were able to produce a factor(s) for the generation of cytolytic activity from L-A-activated T cells. On the other hand, SN from certain antigen-stimulated T cell clones produced factors capable of inducing cytocytic activity by L-A-activated T cells only in the presence of EL-4 SN. Neither EL-4 SN nor cloned T cell SN alone had such a capacity. The nature of the necessary lymphokines in the SN from the clone cells or from the EL-4 is unknown. In the case of the EL-4 SN, it is not known whether the presence of TCGF plays a role or whether that role is perhaps more differentiative than proliferative. This study provides evidence that the induction of CTL from CTL-P can be dissociated into activation, which is required to render T cells responsive to second signals, and differentiation, which is mediated by two different factors.     

In silico model of DSF synthase RpfF protein from Xanthomonas oryzae pv. Oryzae: a novel target for bacterial blight of rice disease

BACKGROUND: Rice plant diseases play a major role as biological constraints on production. One of such rice disease is bacterial leaf blight, caused by Xanthomonas oryzae pv. Oryzae (Xoo). The diffusible signal factor (DSF) synthesized by Xoo has a major role in virulence to rice plant. The DSF synthase RpfF protein, which is related to crotonase superfamily is responsible for the maintaining concentration of DSF. DSF-dependent quorum sensing (QS) system adopts protein- protein interaction mechanism to auto regulates the production of DSF. The antibacterial activity of pesticides against Xoo has not yet been completely understood. Three dimensional structure of RpfF protein was predicted using homology modeling method by MODELLER 9V9 software, SWISS MODEL and GENO3D online tools and structures were validated by Ramachandran plot, TM-Score and RMSD. 3D structure of RpfF (accession number AAL06345) was predicted using DSF synthase of Xanthomonas campestris pv. campestris (Xcc) (PDB ID: 3M6M) as a template. The stereo chemical check reveals the structure developed from the modeller was the best one and the potential ligand binding sites were identified by CASTp Server. The predicted RpfF model provides insight into its structure, active sites and aid in the development of novel inhibitors to control bacterial leaf blight in rice plant. DSF synthase RpfF protein could be used as a novel target to control infection.     

Host DNA Synthesis-Suppressing Factor in Culture Fluid of Tissue Cultures Infected with Measles Virus

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated component which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither UV-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus.     

Detection of glucocorticoid sensitive secretory proteins from human melanoma cells

Summary NEL-M1 human melanoma cells have been established to grow in Ham's F12 medium in the absence of serum, hormones, and exogenous growth factors. Conditioned medium from NEL-M1 cultures stimulates growth of these same cells whereas glucocorticoids retard growth in the presence and absence of conditioned medium. Because recent reports indicate that glucocorticoids inhibit the synthesis of growth factors from a variety of cell types, we hypothesized that glucocorticoids may be inhibiting growth of NEL-M1 cells by either suppressing the synthesis of the autocrine growth factor or regulating other secretory proteins that may inhibit the activity of the autocrine growth factor. Initial studies were done to clearly show that NEL-M1 cells were growth inhibited, both in vivo and in vitro, when exposed to the synthetic glucocorticoid, triamcinolone acetonide (TA). When NEL-M1 cells were injected into nude mice and treated with TA (100 μg per mouse per week) a 67% reduction in tumor mass was observed compared to the control group over a 5-wk growth period. Additional studies show that in a serum-free defined medium TA (100 nM) inhibited growth of MEL-M1 cells by 56% after 6 d in culture. At this same time TA was shown to affect the expression of several proteins secreted from these cells. TA treatment resulted in the appearance of a 125 000 molecular weight protein, suppression of the synthesis-secretion of three proteins (37 000, 57 000, and 76 000 molecular weight) and enhanced expression of a 60 000-molecular weight protein. However when NEL-M1 cells were cultured in conditioned medium from TA-treated cells, a stimulation in both [³H]thymidine incorporation into DNA and cell proliferation was observed. When the conditioned medium was fractionated by Amicon ultrafiltration, the growth stimulatory activity was found in the <10 000 molecular weight fraction. These results demonstrate that glucocorticoids, as a single agent, inhibit the growth of NEL-M1 human melanoma cells. However, this growth inhibition by glucocorticoids may not be through the regulation of the synthesis-secretion of the autocrine growth factor. Furthermore, the data suggest that the glucocorticoid-sensitive secretory proteins may not be directly involved in the in vitro regulation of NEL-M1 cellular growth.