Dexamethasone prevents the growth inhibitory effects of recombinant tumor necrosis factor in a rat hepatoma cell line Reuber-RC-3: an association with the changes in the messenger RNA levels for multidrug resistance gene.

Two days exposure to recombinant tumor necrosis factor (rTNF-alpha) produced a dose-dependent reduction in (methyl-3H) thymidine incorporation in RC-3 cells (ID50 = 25 units/ml). Prolonged treatment with rTNF-alpha further resulted in a significant reduction in colony formation (ID50 = 200 units/ml), which was reversed upon removal of the agent. Interferon levels were undetectable in the supernatants of the rTNF-alpha treated cells. Simultaneous exposure to dexamethasone prevented the growth inhibition in rTNF-alpha-treated RC-3 cells. Significant dose-dependent increase in the steady state levels of the mRNA for multidrug resistance (MDR1) gene was observed after rTNF-alpha treatment while simultaneous exposure to dexamethasone produced a substantial reduction in the mRNA levels for MDR1 gene. These data suggest that growth inhibitory effects of TNF are regulated by dexamethasone and are associated with changes in MDR1 mRNA levels in hepatoma-derived cells.     

The effects of PGD2 and 9-deoxy-Δ9-PGD2 on colony formation of murine osteosarcoma cells

Effects of antineoplastic prostaglandins (PG), PGD₂ and 9-deoxy-Δ⁹-PGD₂, on colony formation of cloned Dunn osteosarcoma (TA 102), normal Swiss 3T3 and V-79 cell lines were evaluated. PGD₂ significantly inhibited the colony formation of TA 102 cells in a dose-dependent manner at concentrations between 0.5 and 5 ug/ml. The IC₅₀ value was calculated to be 0.72 ug/ml. A dose-dependent inhibition of TA 102 colony formation was also observed with 9-deoxy-Δ⁹-PGD₂ between 0.01 to 1 ug/ml, the IC₅₀ value being 0.22 ug/ml. These prostaglandins did not exert cytocidal effects in vitro on Swiss 3T3 cells at concentrations between 0.01 to 1 ug/ml. The two agents had no significant cytocidal effects on V-79 cells except for 9-deoxy-Δ⁹-PGD₂ at a concentration of 5 ug/ml. These results suggest that PGD₂ and 9-deoxy-Δ⁹-PGD₂ are considered to have cytocidal activity on Dunn osteosarcoma cells in dosages which do not affect non-malignant cells.     

The effects of PGD2 and 9-deoxy-Δ-PGD2 on colony formation of murine osteosarcoma cells

Effects of antineoplastic prostaglandins (PG), PGD₂ and 9-deoxy-Δ⁹-PGD₂, on colony formation of cloned Dunn osteosarcoma (TA 102), normal Swiss 3T3 and V-79 cell lines were evaluated. PGD₂ significantly inhibited the colony formation of TA 102 cells in a dose-dependent manner at concentrations between 0.5 and 5 ug/ml. The IC₅₀ value was calculated to be 0.72 ug/ml. A dose-dependent inhibition of TA 102 colony formation was also observed with 9-deoxy-Δ⁹-PGD₂ between 0.01 to 1 ug/ml, the IC₅₀ value being 0.22 ug/ml. These prostaglandins did not exert cytocidal effects in vitro on Swiss 3T3 cells at concentrations between 0.01 to 1 ug/ml. The two agents had no significant cytocidal effects on V-79 cells except for 9-deoxy-Δ⁹-PGD₂ at a concentration of 5 ug/ml. These results suggest that PGD₂ and 9-deoxy-Δ⁹-PGD₂ are considered to have cytocidal activity on Dunn osteosarcoma cells in dosages which do not affect non-malignant cells.     

Morphological differentiation of a murine neuroblastoma clone in monolayer culture induced by dexamethasone

Dexamethasone induces morphological differentiation in murine neuroblastoma cells in culture. The percentage of differentiated cells depends on the concentration of dexamethasone in the medium and duration of treatment. After drug removal (with or without replating), many cells maintain their differentiated phenotype. Dexamethasone-treated cells have larger soma and contain more protein. Dexamethasone also produces a concentration-dependent inhibition of population growth. Growth inhibition is complete by 2 days following treatment with dexamethasone 50 micrograms/ml. Complete growth inhibition is achieved prior to the complete expression of morphological differentiation. Androstenedione, testosterone, and 17-beta-estradiol--all steroids without glucocorticoid activity--have no differentiating effect, although they inhibit growth or cause cell death at higher concentrations.     

The effects of PGD2 and 9-deoxy-delta 9-PGD2 on colony formation of murine osteosarcoma cells

Effects of antineoplastic prostaglandins (PG), PGD2 and 9-deoxy-delta 9-PGD2, on colony formation of cloned Dunn osteosarcoma (TA 102), normal Swiss 3T3 and V-79 cell lines were evaluated. PGD2 significantly inhibited the colony formation of TA 102 cells in a dose-dependent manner at concentrations between 0.5 and 5 micrograms/ml. The IC50 value was calculated to be 0.72 microgram/ml. A dose-dependent inhibition of TA 102 colony formation was also observed with 9-deoxy-delta 9-PGD2 between 0.01 to 1 microgram/ml, the IC50 value being 0.22 microgram/ml. These prostaglandins did not exert cytocidal effects in vitro on Swiss 3T3 cells at concentrations between 0.01 to 1 microgram/ml. The two agents had no significant cytocidal effects on V-79 cells except for 9-deoxy-delta 9-PGD2 at a concentration of 5 ug/ml. These results suggest that PGD2 and 9-deoxy-delta 9-PGD2 are considered to have cytocidal activity on Dunn osteosarcoma cells in dosages which do not affect non-malignant cells.     

Cyclosporine inhibits macrophage-mediated antigen presentation

The influence of cyclosporine on antigen-specific, macrophage-dependent T cell activation was analyzed in vitro. Murine T cell activation by antigens derived from Listeria monocytogenes was monitored by the production of interleukin 2. Pretreatment (2 hr, 37 degrees C) of macrophages with cyclosporine resulted in a cell population with a markedly diminished capacity to support the activation of T lymphocytes. When cyclosporine-pretreated macrophages were added to cultures of untreated T cells and antigen, the dose of cyclosporine that produced 50% inhibition (ID50) was 1.5 micrograms/ml, and if antigen was present during the drug pretreatment, the ID50 was 0.6 micrograms/ml. Pretreatment of T cells also inhibited their subsequent activation by antigen and untreated macrophages, but a higher dose of cyclosporine was required to produce similar inhibition (ID50 = 4.4 micrograms/ml). Additional experiments focused on the mechanism of inhibition of antigen presentation when macrophages were pretreated with the drug. The addition of interleukin 1 or indomethacin to the cultures did not alter the inhibitory effect of cyclosporine. Under conditions that produced greater than 90% inhibition of antigen presentation, macrophage surface Ia expression was not altered, and the uptake and catabolism of radiolabeled antigen remained normal. Thus, cyclosporine had profound effects on antigen presentation that appear to be unrelated to decreases in interleukin 1 production, increases in prostaglandin production, decreases in Ia expression, or changes in antigen uptake and catabolism.     

Zinc levels in zinc-stabilized insulin are inhibitory to the growth of cells in vitro

Adult human prostatic epithelium was cultured in a defined medium consisting of RPMI 1640 supplemented with transferrin, insulin, epidermal growth factor, dexamethasone, and vitamin A. In the presence of insulin, stabilized with zinc, maximum epithelial multiplication was obtained at an insulin concentration of 0.03 to 0.1 U/ml, corresponding to a zinc concentration of 1.4 X 10(-7) M. At higher insulin concentrations, growth stimulation declined. Zinc-free insulin, on the other hand, stimulated cell multiplication with an optimum concentration of 0.3 to 1.0 U/ml. At this concentration, the maximum growth was twice that obtained with zinc-stabilized insulin. Results demonstrate that growth inhibition caused by zinc limits the concentration of zinc-stabilized insulin, which can be used in serum-free, defined culture media.     

Expression of a binding structure for sialic acid-containing glycoconjugates on rat bone marrow-derived macrophages and its modulation by IFN, TNF-alpha, and dexamethasone

Rat macrophages express a binding structure for sialic acid-containing glycoconjugates (sialic acid-binding receptor, SAR) which can be detected by a rosette assay utilizing SRBC coated with bovine brain gangliosides (E-G). Freshly isolated rat bone marrow cells (BMC) contain about 5% SAR-positive cells. Rat BMC cultured for 1 wk with tissue culture media containing CSF-1 differentiate into a virtually pure population of bone marrow-derived macrophages (BMDM phi). All BMDM phi bound E-G coated with an optimal concentration of gangliosides (100 micrograms/ml). When BMC were cultured for 1 wk with murine recombinant granulocyte-macrophage CSF, irrespective of the dose of GM-CSF, approximately 90% of the cells were identified as rat macrophages, and practically all expressed SAR. Only about 50% of BMDM phi bound SRBC coated with a suboptimal concentration of gangliosides (20 micrograms/ml). However, this percentage increased markedly after 8 to 72 h incubation with 1 to 10,000 U/ml purified murine IFN-alpha or IFN-beta, whereas murine or rat rIFN-gamma at doses above 10 U/ml led to a decrease of E-G binding. Human and murine rTNF-alpha enhanced rosette formation in a dose-dependent manner. These effects could be blocked by the respective anti-cytokine antibodies. Treatment of BMDM phi with dexamethasone also augmented E-G rosetting. The enhancement of E-G binding was abolished by pretreatment of BMDM phi with cycloheximide and actinomycin D but not with mitomycin C, suggesting that de novo synthesis of protein and RNA, but not DNA, is required. Our results demonstrate that all rat BMDM phi constitutively bear SAR, and that murine IFN-alpha, IFN-beta, and TNF-alpha, as well as dexamethasone, may augment SAR expression.     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.     

Comparison of two dosages of prostaglandin F2a on canine uterine motility

The effect of 50 ug/kg prostaglandin F2a (PGF2a) was compared with 250 ug/kg PGF2a on uterine motility in the diestrous female. Microtipped pressure transducers were surgically implanted in the uteri of 6 females at 30 days diestrus and in 6 females at 60 days diestrus. Uterine responses to intravenous PGF2a (5 ug/kg), oxytocin (0.05 USP units/kg), and intramuscular PGF2a (50 ug/kg and 250 ug/kg) were measured in the awake females on Days 1 and 2 after implantation. There was no significant difference in the increase in intrauterine pressure produced by 50 ug/kg of PGF2a compared with 250 ug/kg of PGF2a. The longest duration of the effect occurred when 250 ug/kg of PGF2a were given. Side effects were also documented. Significantly more vomiting occurred when 250 ug/kg PGF2a were given than when 50 ug/kg PGF2a were administered. The only advantage to using a higher dosage of PGF2a appears to be the longer duration of motility.     

Inhibition of protein synthesis by a toxic lectin from Viscum album L. (mistletoe).

1. The haemagglutinating and toxic lectin from Viscum album L. (mistletoe) inhibits protein synthesis in a lysate of rabbit reticulocytes, with an ID50 (concentration giving 50% inhibition) of 2.6 microgram/ml. This effect is enhanced (ID50 0.21 microgram/ml) if the lectin is reduced with 2-mercaptoethanol. 2. The lectin inhibits protein synthesis also in BL8L cells in culture. Inhibition occurs after a lag time of 3 h. The ID50 is 7 ng/ml, and increases after reduction of the lectin. 3. This and the gross lesions observed in rats poisoned with V. album lectin indicate this is a toxin very similar to ricin.     

The anti-chlamydial and anti-proliferative activities of recombinant murine interferon-gamma are not dependent on tryptophan concentrations

The effect of recombinant murine interferon-gamma on the growth of Chlamydia trachomatis was analyzed in a mouse fibroblast cell line (McCoy cells). Murine interferon-gamma had a very potent anti-chlamydial activity, although minimally affecting cellular proliferation. Over 95% inhibition of chlamydial inclusions was obtained at a concentration of 1 U/ml of interferon. At a concentration of 1 U/ml of murine interferon-gamma, there was minimal inhibition of the proliferative capacity of McCoy cells. Approximately 50% inhibition of cell growth was obtained with a concentration of 10 U/ml of interferon. Varying concentrations of tryptophan in the medium did not alter either the anti-chlamydial or the anti-proliferative activity of the interferon.     

Inhibition of deoxyribonucleic acid repair in Escherichia coli by caffeine and acriflavine after ultraviolet irradiation.

The effects of caffeine and acriflavine on cell survival, single-strand deoxyribonucleic acid break formation, and postreplication repair in Escherichia coli wild-type WP2 and WP2 uvrA strains after ultraviolet irradiation was studied. Caffeine (0.5 mg/ml) added before and immediately after ultraviolet irradiation inhibited single-strand deoxyribonucleic acid breakage in wild-type WP2 cells. Single-strand breaks, once formed, were no longer subject to repair inhibition by caffeine. At 0.5 to 2 mg/ml, caffeine did not affect postreplication repair in uvrA strains. These data are consistent with the survival data of both irradiated WP2 and uvrA strains in the presence and absence of caffeine. In unirradiated WP2 and uvrA strains, however, a high caffeine concentration (greater than 2 mg/ml) resulted in gradual reduction of colony-forming units. At a concentration insufficient to alter survival of unirradiated cells, acriflavine (2 microgram/ml) inhibited both single-strand deoxyribonucleic acid breakage and postreplication repair after ultraviolet irradiation. These data suggest that although the modes of action for both caffeine and acriflavine may be similar in the inhibition of single-strand deoxyribonucleic acid break formation, they differ in their mechanisms of action on postreplication repair.     

Inhibiting action of anti-arrhythmia agents of the phenothiazine series--etmozin and its diethylamino analog--on platelet aggregation and metabolism of endogenous arachidonic acid

Effect of the cardiotropic drugs of the phenothiazine series ethmozine, and its diethylamine analogue (DAAE), on platelet aggregation and formation of arachidonic acid metabolites has been studied. Both drugs inhibit the ADP-induced aggregation in the platelet-rich plasma. Ethmozine inhibits only the second (irreversible) wave of aggregation, while DAAE inhibits both the first (reversible) and the second one. 50% inhibition (ID50) of the second wave of aggregation is observed at the following concentrations of the two agents: 300-500 micrograms/ml (ethmozine) and 20 micrograms/ml (DAAE). DAAE completely inhibits the irreversible aggregation of platelets washed off plasma, induced by arachidonic acid (ID50 approximately 30 micrograms/ml) and Ca2+-ionophore A23187 (ID approximately 55 micrograms/ml); the aggregation, induced by thrombin is inhibited by 80-90% (ID approximately 130 micrograms/ml). Formation of arachidonic acid metabolites in platelets effected by these inducers was measured by the accumulation of malondialdehyde (MDA). DAAE fails to inhibit MDA formation induced by exogenous arachidonic acid, but completely prevents the synthesis of MDA induced by A23187 and thrombin. These data suggest that DAAE inhibits the release of endogenous arachidonic acid from membrane phospholipids catalysed by phospholipase A2, but does not affect its subsequent metabolic transformations. In all probability, ethmozine and DAAE, just as other phenothiazines, affect platelets via the inhibition of Ca2+-calmodulin-dependent reactions and processes.     

Effects of recombinant human tumor necrosis factor (rhTNF) on normal human and mouse hemopoietic progenitor cells

We examined the effects of recombinant human tumor necrosis factor (rhTNF) on normal human and murine granulocyte-macrophage (CFU-gm) and erythroid (CFU-e, BFU-e) progenitor cells. We suppressed in vitro colony formation by human marrow CFU-gm, CFU-e and BFU-e or peripheral blood BFU-e by adding rhTNF to the culture in a dose-related manner. A half-maximal inhibition was observed with 1-10 ng/ml. Leukemic cell line K562 cells were found to be sensitive to rhTNF in the clonogenic colony assay. However, the clonal growth of murine marrow CFU-e and BFU-e colonies was less than 50% inhibited and CFU-gm growth was unaffected even at a concentration of 1,000 ng/ml. We observed slight to moderate inhibition after 24 h pulse exposure of both human and murine-committed progenitors to rhTNF prior to the culture. Intravenous injection of 1 mg/kg of rhTNF caused a marked decrease in marrow erythroid progenitors and consequently caused anemia in the mice. Our data indicate that rhTNF has a suppressive effect on normal human and murine hemopoietic colony formation in vitro and murine erythropoiesis in vivo.     

Zinc levels in zinc-stabilized insulin are inhibitory to the growth of cells in vitro

Summary Adult human prostatic epithelium was cultured in a defined medium consisting of RPMI 1640 supplemented with transferrin, insulin, epidermal growth factor, dexamethasone, and vitamin A. In the presence of insulin, stabilized with zinc, maximum epithelial multiplication was obtained at an insulin concentration of 0.03 to 0.1 U/ml, corresponding to a zinc concentration of 1.4×10⁻⁷ M. At higher insulin concentrations, growth stimulation declined. Zinc-free insulin, on the other hand, stimulated cell multiplication with an optimum concentration of 0.3 to 1.0 U/ml. At this concentration the maximum growth was twice that obtained with zinc-stabilized insulin. Results demonstrate that growth inhibition caused by zinc limits the concentration of zinc-stabilized insulin, which can be used in serum-free, defined culture media. This work was supported by the Division of Cancer Cause and Prevention, National Cancer Institute, DHHS Grant No. CA-28279 to Webber.     

Effects of recombinant human interferon alpha on human megakaryocyte and fibroblast colony formation

Effects of recombinant human interferon alpha (HuIFN-alpha) on human megakaryocyte (CFU-MK) and fibroblast (CFU-F) colony-forming cell growth were studied. Concentration-dependent inhibition of both CFU-MK and CFU-F by HuIFN-alpha was demonstrated. Statistically significant suppression of both CFU-MK and CFU-F was seen at a HuIFN-alpha concentration of 1000 U/ml or greater. No significant difference was found between HuIFN-alpha treated cultures and controls for the distribution of CFU-MK types and for the size and cell morphology of CFU-F. When a concentration of 1000 u/ml HuIFN-alpha was added at varying time points during the marrow cultures, decreased numbers of megakaryocyte and fibroblast colonies only appeared at the early days of cultures. When bone marrow cells were incubated with HuIFN-alpha for different periods of time prior to initiation of cultures, a reduction of megakaryocyte colony formation also occurred. These studies demonstrate a suppressive effect of HuIFN-alpha on human CFU-MK and CFU-F growth. This effect seems to occur at the initial stages of CFU-MK and CFU-F development.     

Synthesis and antiproliferative activity in vitro of novel (2-butynyl)thioquinolines

The series of new acetylenic thioquinolines containing propargyl, 2-butynyl, 4-bromo-2-butynyl, and 4-hydroxy-2-butynyl groups has been prepared and tested for antiproliferative activity in vitro against human [SW707 (colorectal adenocarcinoma), CCRF/CEM (leukemia)] and murine [P388 (leukemia), B16 (melanoma)] cancer lines. All the compounds obtained exhibited antiproliferative activity. The most active compounds 7, 16, 17, and 19 have the ID(50) values ranging from 0.2 to 4.6 microg/ml comparable to that of cisplatin used as reference compounds.     

Basic fibroblast growth factor (B-FGF) induces early- (CFU-s) and late-stage hematopoietic progenitor cell colony formation (CFU-gm, CFU-meg, and BFU-e) by synergizing with GM-CSF, Meg-CSF, and erythropoietin, and is a radioprotective agent in vitro

Basic fibroblastic growth factor (B-FGF) is a hormone-like protein which belongs to a class of heparin-binding growth factors. B-FGF is synthesized and released to circulate in the blood where it can be recognized by target cells through specific high-affinity plasma membrane receptors. B-FGF is known to be a potent mitogen for a number of specific cell types. We report data which demonstrates B-FGF can influence noncommited and specific lineage-derived hematopoietic progenitors when incubated in vitro. When combined with adherent cell-depleted normal murine marrow cells, B-FGF increased the number of both day 9 and day 12 spleen colony-forming units (CFU-s) from lethally irradiated animals. However, day 12-derived CFU-s were more sensitive to B-FGF, since optimal CFU-s production was observed at 10 ng/ml vs. 100 ng/ml for day 9 CFU-s (p less than 0.05). In adherent cell-depleted murine and human marrow cultures, the addition of B-FGF possessed synergistic activity in combination with the optimal concentration of GM-CSF for CFU-gm at a dose of 10 ng/ml which was inhibited in the presence of protamine sulfate (LD50 dose, 100 mu gm/ml), an inhibitor of B-FGF mitogenic activity, or in the presence of heparin (LD50 dose, 100 U/ml), an effective B-FGF binding agent. B-FGF also expressed synergistic activity in the presence of optimal concentrations of erythropoietin and Meg-CSF for murine and human BFU-e, and murine CFU-meg. No in vitro colony formation was observed when cells were cultured in the presence of B-FGF, but in the absence of the specific hematopoietic growth factor. Finally, B-FGF was also shown to be an effective radioprotective agent in vitro. Murine and human CFU-gm exposed to increasing doses of radiation (0.5 to 5 Gy) combined with GM-CSF and increasing doses of B-FGF (0.1 to 100 ng/ml) produced less radiation-induced toxicity compared to cultures containing GM-CSF alone. This data demonstrates B-FGF influences early- and late-stage hematopoietic progenitors, possesses synergistic activity with hematopoietic growth factors, and is a radioprotective agent in vitro. These results suggest B-FGF must be considered as a member of the family of molecules capable of influencing hematopoiesis in vitro.