Mechanism of erythropoietin action on the erythroid progenitor cells induced from murine erythroleukemia cells (TSA8)

Erythropoietin is a well-known erythroid differentiation and growth factor, but the mechanism of its action is not well understood. In this work, we have examined its mechanism of action on the erythropoietin-responsive murine erythroleukemia cells (TSA8). TSA8 cells become responsive to erythropoietin after induction with DMSO. Stimulatory effects on erythropoietin response are observed with the addition of compounds affecting the cAMP level such as forskolin, phosphodiesterase inhibitor and cholera toxin only in the presence of erythropoietin. cAMP analogues themselves show no stimulatory effect on TSA8 cells, nor does erythropoietin increase cAMP level in the cells. Thus, it is suggested that cAMP does not act as a direct second messenger for signal transduction through erythropoietin receptors, but as a stimulator of the erythropoietin receptor pathway and/or as a second messenger in combination with the receptor pathway. The mechanism for acquisition of responsiveness to growth and differentiation factors of progenitor cells is discussed.     

Characteristics of cyclic AMP enhancement of retinoic acid induction of increased transglutaminase activity in HL60 cells

When the human myeloid leukemia cell line (HL60) is induced to differentiate with retinoic acid (RA), there is a concentration-dependent increase in transglutaminase (TGase) activity which peaks on day 5. While dibutyryl 3',5'-cyclic adenosine monophosphate (db-cAMP) alone produced only a slight increase in TGase activity in HL60 cells, the concomitant addition of db-cAMP (100 microM) with RA (10(-12)-10(-4) M) potentiates RA induction of TGase activity. Maximal increases in TGase activity (2- to 10-fold) were observed with 10(-4)-10(-7) M RA and when db-cAMP was present from 24 to 48 h after the addition of RA. The cyclic nucleotide enhancement was dose-dependent from 10 to 100 microM of cAMP. Less marked increases were observed with 8-bromo-cAMP and with the phosphodiesterase inhibitor theophylline. Although the simultaneous addition of PGE1 or PGE2 (10(-8)-10(-6) M) produced no enhancement of RA-induced TGase activity, adding PGE1 or PGE2 24 or 48 h following RA treatments produced an enhancement of TGase activity. The phosphodiesterase inhibitor potentiated the increases produced by db-cAMP and the prostaglandins. Dibutyryl cAMP enhanced the ability of RA to induce the cells to reduce nitroblue tetrazolium (NBT), a functional measure of differentiation, at lower concentrations of RA and with shorter treatment durations. cAMP potentiates RA-induced TGase activity in HL60 cells and the combination appears to be associated with enhanced RA-induced differentiation.     

Regulation of Sertoli cell cyclic adenosine 3':5' monophosphate phosphodiesterase activity by follicle stimulating hormone and dibutyrl cyclic AMP

Total phosphodiesterase activity was measured in Sertoli cell culture after exposure to isobutyl-methyl-xanthine, dibutyryl cyclic AMP and FSH. After 24 hr of incubation both FSH and dibutyryl cAMP caused a significant increase in total phosphodiesterase activity of Sertoli cell homogenates (control: 66 ± 16 pmoles/min/mg protein; FSH: 291 ± 25 pmoles/min/mg protein; dibutyryl cAMP: 630 ± 70 pmoles/min/mg protein). FSH stimulation was potentiated by isobutyl-methyl-xanthine. Both in the presence and absence of xanthine, the induction of phosphodiesterase was dependent on the FSH concentration, with maximal stimulation achieved with 0.5–1.0 μg FSH/ml. The induction of phosphodiesterase activity by hormone was abolished by cycloheximide treatment. The data suggest that FSH regulates phosphodiesterase activity via changes of cAMP levels in Sertoli cell in culture.     

Theophylline Potentiates Lipopolysaccharide-Induced NO Production in Cultured Astrocytes

Elucidation of the functions of astrocytes is important for understanding of the pathogenic mechanism of various neurodegenerative diseases. Theophylline is a common drug for bronchial asthma and occasionally develops side-effects, such as acute encephalopathy; although the pathogenic mechanism of the side-effects is unknown. The lipopolysaccharide (LPS)-induced nitric oxide (NO) production is generally used for an index of the activation of astrocyte in vitro. In this study, in order to elucidate the effect of theophylline on the astrocytic functions, we examined the LPS-induced NO production and the expression of iNOS in cultured rat cortex astrocytes. Theophylline alone could not induce the NO production; however, NO production induced by LPS was enhanced by theophylline in a dose-dependent manner; and by isobutylmethylxanthine, a phosphodiesterase inhibitor. The theophylline enhancement of LPS-induced NO production was further increased by dibutyryl cyclic AMP, a membrane-permeable cAMP analog; and by forskolin, an adenylate cyclase activator. When the cells were preincubated with Rp-8-Br-cAMP, an inhibitor of protein kinase A, the theophylline enhancement of LPS-induced NO production was decreased. The extent of iNOS protein expression induced by LPS was also enhanced by theophylline. It is likely that phosphodiesterase inhibition is a major action mechanism for the theophylline enhancement of LPS-induced NO production in astrocytes. Theophylline-induced acute encephalopathy might be due to the hyper-activation of astrocytes via cAMP signaling to produce excess amount of NO.     

Cyclic AMP and the vascular action of parathyroid hormone

The involvement of tissue cAMP in the vasodilating action of parathyroid hormone (PTH) was investigated. The bovine active fragment bPTH-(1-34) was used in all studies. In anesthetized dogs, theophylline, a phosphodiesterase inhibitor, potentiated the hypotensive action of bPTH-(1-34) at the dose of 1 microgram/kg. The potentiation was related to the dose of theophylline infused. In an in vitro rat tail artery helical strip assay, dibutyryl cAMP produced dose-related relaxation in arginine vasopressin (AVP) constricted blood vessels. bPTH-(1-34) also produced dose-related relaxation in the tail artery constricted by AVP. In the presence of isobutylmethylxanthine, another phosphodiesterase inhibitor, the bPTH-(1-34) dose--response curve was shifted to the left, indicating potentiation. Imidazole, which has phosphodiesterase stimulating activity, significantly decreased the in vitro vasorelaxing effect of bPTH-(1-34). In addition, bPTH-(1-34) increased significantly the rat tail artery cAMP content. b-PTH-(1-34) oxidized with hydrogen peroxide lost its vasorelaxing activity and was also ineffective in increasing the tail artery cAMP content. All these data strongly suggest that cAMP may be involved in eliciting the vasorelaxing action of bPTH-(1-34).     

Stimulation of bovine endothelial cell angiotensin-I-converting enzyme activity by cyclic AMP-related agents

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of cyclic nucleotides, isoproterenol (beta adrenergic agonist), and theophylline (phosphodiesterase inhibitor) on angiotensin-I-converting enzyme (ACE) activity of the cells and culture medium. Dibutyryl cAMP (10(-3) M) but not cAMP or dibutyryl cGMP stimulated angiotensin-I-converting enzyme (ACE) activity of cells in culture approximately 50-100% but had little influence on ACE activity of the medium. Theophylline at 10(-3) M concentration produced a three- to fourfold stimulation of both cellular and medium ACE activity. Isoproterenol by itself had no effect on cellular ACE activity but produced a stimulatory effect at 10(-7)-10(-5) M concentration after pretreatment of cells for 24 hr with 10(-4) M theophylline. The results support the concept that ACE activity of endothelial cells is influenced by the cyclic AMP system. ACE activity in cells and that released into medium may be under different regulatory controls.     

Modulation by cAMP of 1alpha,25-dihydroxyvitamin D3 sensitivity of murine erythroleukemia cells.

As we previously reported, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently inhibited not only proliferation of undifferentiated murine erythroleukemia (MEL) cells but also activin A-induced erythroid differentiation of MEL cells. However, the effect of 1,25(OH)2D3 on MEL cell proliferation was significantly greater by one order of magnitude than that on differentiation (IC(50): 9.2 vs 0.8 nM, respectively). The response of activin A-treated mature MEL cells to 1,25(OH)2D3 in the induction of 1,25(OH)2D3-24-hydroxylase (24-OHase) activity, a rapid effect of 1,25(OH)2D3, was enhanced to the same degree as in untreated immature cells, suggesting that differences in capacity of cells to inactivate 1,25(OH)2D3 did not contribute to augmentation of 1,25(OH)2D3 effect in activin A-treated mature cells. Furthermore, neither the number nor the affinity of vitamin D receptors (VDR) differed significantly between activin A-treated cells and untreated immature cells. The intracellular cAMP level, which affects 1,25(OH)2D3-mediated induction of 24-OHase activity, was significantly less in activin A-treated mature cells than in immature MEL cells. The addition of dibutyryl cAMP (dbc AMP) to activin A-treated MEL cells dose-dependently attenuated 1,25(OH)2D3-mediated induction of 24-OHase activity, finally to a level comparable to that of the untreated cells at the final concentration of 100 nM dbcAMP, while dbcAMP itself by 100 nM did not affect MEL cell differentiation by 24 h. In summary, we have shown for the first time that 1,25(OH)2D3 exerted its effect on leukemia cells at physiological concentration and that the magnitude of this effect depended on the changes in intracellular cAMP level through stages of differentiation, suggesting that the cAMP-protein kinase A system may be useful as a target for clinical application of vitamin D analogs by improving the sensitivity of leukemic cells to 1,25(OH)2D3.     

Autocrine differentiation-inhibiting factor (ADIF) from chicken erythroleukemia cells acts on human and mouse early BFU-E erythroid progenitors

tsAEV-LSCC HD3 chicken erythroid cells transformed by the avian erythroblastosis virus (AEV) secrete an autocrine differentiation-inhibiting factor, ADIF, which blocks differentiation without affecting proliferation of the chicken erythroid cells that synthesize and secrete it into the culture medium. The chicken erythroleukemia cell ADIF activity is not restricted to avians. It prevents dimethylsulfoxide (DMSO) from stimulating murine Friend erythroleukemia cells to synthesize hemoglobin. ADIF also blocks erythroid differentiation in normal human and murine bone marrow where it selectively targets the early BFU-E (burst-forming) erythroid precursor cells without affecting the more advanced CFU-E erythroid precursor cells or cells of the different granulocyte-macrophage lineage.     

Differential actions of phosphodiesterase inhibitors on secretin- and vasoactive intestinal peptide-induced increases in chief cell cAMP

The actions of three different phosphodiesterase inhibitors, theophylline, 3-isobutyl-1-methylxanthine (IBMX) and Ro 20-1724 (Ro), on cellular cAMP and pepsinogen secretion from dispersed chief cells prepared from guinea pig stomach were examined. The relative order of potency for increasing cAMP and pepsinogen secretion was Ro greater than IBMX greater than theophylline. Ro, the most efficacious agent, caused a 17-fold increase in basal cAMP and a similar augmentation of the increase in cAMP caused by secretin or vasoactive intestinal peptide (VIP). Differential actions of these agents on the dose-response curves for secretin- and VIP-induced increases in cAMP suggest that chief cell receptors for these peptides are coupled to pools of cAMP that are acted upon by heterogeneous phosphodiesterases with varying sensitivities to inhibitors. Moreover, Ro, a selective inhibitor of low Km cAMP-specific phosphodiesterases, is the most potent and efficacious agent tested in this cell system.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

Possible role of H1 histone in the differentiation of mouse erythroleukemia cells

Serum-starved mouse erythroleukemia cells, stationary phase cells or cells cultured in dibutyryl cAMP (1 mM) can be induced to differentiate by addition of 20% fetal calf serum plus cycloheximide. Culturing unstarved log phase cells in 20% fetal calf serum plus low levels of cycloheximide and histone H1 also causes a significant level of differentiation. These same concentrations of cycloheximide and H1 histone employed separately with 20% fetal calf serum do not induce differentiation. The role these procedures may have in causing an accumulation of histone H1 and cell differentiation is discussed.     

Activation of latent EBV via anti-IgG-triggered, second messenger pathways in the Burkitt's lymphoma cell line Akata

Anti-IgG treatment activated latent EBV genomes in 50 to 70% of the cells of the Burkitt's lymphoma cell line Akata. The EBV-activating role of intracellular Ca2+, as potentiated by diacylglycerol (DAG) and suppressed by cAMP, was analyzed in the cells through effects of agonists and antagonists of these second messenger pathways. Early Ag (EA) was induced in 10% of cells with the calcium ionophore A23187 (A23187). EA induction with anti-IgG or A23187 was blocked by a calmodulin antagonist, trifluoperazine. The DAG pathway had a potentiating but not direct effect on EBV activation because: 1) the DAG analog, dioctanoylglycerol (diC8), an agonist for protein kinase C, alone induced only 2% EA-positive cells, 2) diC8 synergized with A23187 for EA induction, and 3) the protein kinase C antagonist, staurosporine, almost completely inhibited EA induction by anti-IgG. When cells were reincubated in medium with fresh diC8 and A23187 at 3, 6, 9, and 12 h, EA induction at 24 h reached the levels seen with anti-IgG stimulation. A cAMP-mediated pathway suppressed EBV activation because dibutyryl cAMP or 8-bromo-cAMP, plus blockage of phosphodiesterase by theophylline, or use of forskolin, inhibited EA induction with anti-IgG. Although the principal stimulatory role in EBV activation of a Ca2(+)-mediated, second messenger pathway, as synergized by DAG and inhibited by cAMP, was established, we did not explain the significant lag in EA induction by A23187 and diC8 as compared with anti-IgG induction of EA. We conclude that EBV genome activation with anti-IgG is mediated by Ca2+/calmodulin and DAG pathways in Akata cells, that the cAMP pathway suppresses EA induction by anti-IgG, and that a mechanism regulating the speed of EA induction remains unexplained.     

Melanocyte-stimulating hormone affects melanogenic differentiation of quail neural crest cells in vitro

Quail neural crest cells were treated in vitro with alpha-melanocyte-stimulating hormone (alpha-MSH) or dibutyryl cyclic AMP (dbcAMP) plus theophylline. These treatments increased the proportion of melanocytes to total cells in crest cell outgrowth cultures. Pigmentation of neural crest cell clusters proceeded more rapidly when cultures were treated with alpha-MSH or dbcAMP plus theophylline than when untreated. In clonal cell cultures, the proportion of pigmented colonies to total colonies was increased by MSH treatment. From these results, MSH seems not only to accelerate melanogenic differentiation but also to affect the state of commitment of neural crest cells to melanogenic differentiation in vitro, and this action of MSH appears to be mediated by cAMP.     

The role of butyrate in the reverse transformation reaction in mammalian cells.

The reverse transformation reaction of Chinese hamster ovary cells from compact, epithelial-like, randomly growing, heavily knobbed, lectin reactive cells into stretched, tighly adherent, smooth-surfaced, lectin resistant, fibroblast-like cells normally elicited by dibutyryl cAMP can be produced to its complete extent by N6-monobutyryl cAMP or 8-bromo-cAMP, O2'-monobutyryl cAMP is ineffective as is cAMP itself in the absence of an inhibitor of phosphodiesterase activity. In the presence of a phosphodiesterase inhibitor, cAMP is fully effective. These results indicate that the role of the butyryl groups of dibutyryl cAMP and, especially, the N6-butyryl, in the reverse transformation reaction is protection of the cAMP analogue from degradation. Butyrate at concentrations of about 1 mM does produce a response which to some extent mimics that of cAMP analogues. The cells, however, fail to assume a fibroblastic-like shape, but rather become flattened. The butyrate effect is much slower and less readily reversible than that evoked by cAMP analogues. Butyrate produces an approximately 2-fold increase in intracellular cAMP levels. These results are consistent with the hypothesis that butyrate effects, in part, are mediated by AMP.     

Cyclic AMP-Elevating Agents Inhibit Proliferation of Keratinizing Guinea Pig Epidermal Cells

Cultured guinea pig epidermal cells and dermal fibroblasts were chosen as model systems to study possible growth inhibition by cyclic AMP (cAMP)-elevating drugs. The rate of DNA synthesis was used to assay growth rate in control cultures and those treated with agents which increase intracellular cAMP, including dibutyryl cAMP, the phosphodiesterase inhibitors papaverine and theophylline and agents which stimulate adenylate cyclase, iso-proterenol and prostaglandin E₂ methyl ester. Treatment for 24 h with dibutyryl cAMP (10⁻⁴ to 10⁻² M) inhibited cell growth by 50 to 95%, whereas butyrate(10⁻⁴M) showed essentially no effect. This inhibition could not be attributed to decreased precursor transport or to drug toxicity. Papaverine (10⁻⁶ to 10⁻⁴ M) and theophylline (10⁻⁴ to 10⁻³ M) also gave dose-dependent growth inhibition as did isoproterenol and prostaglandinE₂methyl ester. Radioautographic analysis of grain density after dibutyryl cAMP treatment and ³H-thymidine incorporation indicated no S-phase inhibition. Cyclic AMP-elevating drugs appear to inhibit growth of guinea-pig epidermal cells and dermal flbroblasts by blocking the cell cycle in G₋₂, M₁, or G. ₋₁     

Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin E1

BL191, a newly developed phosphodiesterase inhibitor, markedly potentiated a differentiation of neuroblastoma cell clones (Neuro2a, NS-20Y, and N1E115) induced by dibutyryl cyclic adensoine 3′:5′-monophosphate(dibutyryl cAMP) and prostaglandin E₁ (PGE₁). BL191 (1 mM) inhibited DNA synthesis more strongly when used together with PGE₁ (0.5 μg/ml) and dibutyryl cAMP (0.5 mM) than papaverine (1.6 μg/ml) alone did. The inhibition rates of DNA synthesis were 72.5% for N1E-115, 75.3% for Neuro2a, and 82.5% for NS-20Y. After the treatment with BL191. PGE₁, and dibutyryl cAMP for 48 h all of three cell lines became enlarged and flattened, and extended long processes. The specific activities of choline acetyl transferase (EC 2.3.1.9) of NS-20Y and dopamine β-hydroxylase (EC 1.14.17.1) of N1E-115 increased about 3-fold as compared to the controls. The tumorigenicities of Neuro2a and N1E-115 cells were decreased, but not of NS-20Y. These data suggest the heterogenous responsiveness in neuroblastoma cells to drug treatment.     

Inhibition of maturation and metabolism in rat oocytes by cyclic amp.

It is known that dibutyryl cyclic AMP (dbcAMP) and theophylline inhibit the spontaneous maturation of isolated mouse oocytes. The present study demonstrates that dbcAMP (0.01-1.0 mM) as well as cyclic AMP (cAMP, 10 mM) and a phosphodiesterase inhibitor (IBMX, 0.01-1.0 mM) prevent spontaneous maturation of isolated rat oocytes. As reported earlier an increase in oxygen consumption by the oocyte was found following maturation. When the oocytes were cultured in the presence of dbcAMP or cAMP no change in respiration occurred during culture. These results argue against the theory that cAMP acts as a direct mediator of the action of luteinizing hormone (LH) on oocyte maturation. Furthermore they suggest that changes in oocyte energy metabolism are closely related to the maturation process.     

Cyclic AMP induces activity increase of kinase FA (a transmembrane signal of insulin) during NG108-15 hybrid cell differentiation

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) has been identified to exist in neuroblastoma x glioma hybrid 108-15 cells (NG108-15 cells). More importantly, when NG cells were induced to differentiate with N6, O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), the cellular activity of kinase FA was found to increase dramatically. Time course study further revealed that induction of differentiation in NG cells by dibutyryl cAMP treatment increased the FA activity to over 3 times the levels found in undifferentiated cells and in a linear day-dependent manner, indicating that the FA activity level is correlated with the state of differentiation of NG108-15 cells. This is the first report providing initial evidence that protein kinase FA (a transmembrane signal of insulin) is involved in the induction of neuronal cell differentiation.     

Developmental program of murine erythroleukemia cells. Effect of the inhibition of protein synthesis

The relationship between protein synthesis and commitment to terminal erythroid differentiation by dimethylsulfoxide-treated murine erythroleukemia (MEL) cells has been studied. Treatment with cycloheximide blocks the commitment of MEL cells. The effects of cycloheximide are completely reversible, however. Treatment of MEL cells before commitment delays commitment for a period of time equal to the length of inhibitor treatment. Puromycin exerts a similar effect on the commitment of MEL cells. These results indicate that there is a continuous requirement for protein synthesis before the commitment event.