Alteration in gene expression at the onset of human Y-79 retinoblastoma cell differentiation

We have tested the hypothesis that differentiation and growth arrest of Y-79 human retinoblastoma cells in culture is associated with a modification of gene expression. We first examined proteins translated from mRNAs isolated from Y-79 cells growing in suspension and in attachment cultures in serum-containing medium and found them to be markedly different. This suggests that membrane-substrate interactions are of major consequence in the biochemical differentiation of these cells. Secondly, we examined the patterns of proteins translated from attached cells which had been induced to morphologically differentiate into neuronal-like and glial-like cells by serum-withdrawal and dibutyryl cAMP treatment respectively. The in vitro translatable proteins of mRNAs isolated from these cultures were found to be markedly different from those of the suspension and attachment cultures. Thirdly, we found that treatment of cells growing in attachment culture in serum-containing medium supplemented with 8-bromo cAMP, butyrate and retinoic acid as well as dibutyryl cAMP resulted in discreet alterations in proteins translated in vitro from extracted mRNAs. Although all these substances inhibit the growth of Y-79 cells, only dibutyryl cAMP and butyrate result in morphological differentiation of cells. Our results suggest that (1) attachment and morphological differentiation of Y-79 cells are both related to specific alterations in gene expression and (2) differentiation and inhibition of cell growth by various agents can be correlated with changes in translatable mRNA species although all agents do not act in the same mode.     

Reversible effects of dibutyryl cAMP on laminin and type IV collagen secretion from retinoic acid treated F9 cells

The effects of dibutyryl cAMP on the differentiation of embryonal carcinoma F9 cells were studied mainly using the secretion of laminin and type IV collagen as the marker. For this purpose, F9 cells were labeled with 35S-methionine and radioactive proteins in the medium were analyzed by SDS-polyacrylamide gel electrophoresis. Treatment of F9 cells with retinoic acid alone induced differentiation into cells secreting type IV collagen. The combination of retinoic acid and dibutyryl cAMP stimulated laminin secretion in addition to type IV collagen secretion. This effect of dibutyryl cAMP was observed only 16 h after adding dibutyryl cAMP. Immunofluorescence staining demonstrated that the majority of the cells in culture were converted into cells secreting laminin under these conditions. In contrast to the irreversible effect of retinoic acid, the effect of dibutyryl cAMP on laminin and type IV collagen secretion was reversible at least during the first 5 days of maintaining cells in the medium containing retinoic acid plus dibutyryl cAMP. Removal of dibutyryl cAMP from the culture medium decreased the protein secretion to the basal levels within 2 days. This reversibility was not due to a change in cell number. An in vitro translation assay also suggested the reversible effect of dibutyryl cAMP on the levels of laminin mRNA. Coinciding with variations of the protein secretion, a reversible and homogeneous change in the morphology of retinoic acid generated F9 cells was observed by dibutyryl cAMP.     

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.     

Inhibition of chondrogenesis by retinoic acid in limb mesenchymal cells in vitro: Effects on PGE2 and cyclic AMP concentrations

Effects of retinoic acid (RA) on prostaglandin E₂ (PGE₂) and cyclic AMP (cAMP) concentrations were investigated in high density, micromass cultures of mesenchymal cells derived from chick limb buds. Exposure of cells during the initial 24 h of culture to RA concentrations between 0.05–1.0 μg/ml inhibited chondrogenesis in a dose-dependent manner with 1.0 μg/ml totally inhibiting cartilage formation. Concentrations of PGE₂ and cAMP increased during the prechondrogenic period in control cells in a closely related way and remained elevated throughout the six-day period examined. Addition of RA (0.05 and 0.5 μg/ml) did not significantly alter cAMP concentrations at any time point, but significantly elevated PGE₂ levels relative to control cells in six-day cultures in a concentration-dependent manner. Addition of dibutyryl cAMP enhanced chondrogenesis in control cells between days 3 and 4, but failed to alter the inhibitory effect of RA on chondrogenesis. The results indicate that while PGE₂ and cAMP are important signals in cartilage differentiation, the inhibitory effects of RA on this process are mediated through some other mechanism.     

Separation of mitogen-induced suppressor and helper cell activities during inhibition of interferon production by cyclic AMP.

The effect of exogenous cyclic AMP on mitogen-induced suppression and enhancement of the in vitro plaque-forming cell (PFC) response and on mitogen induction of immune interferon (also called type II) in cultures was examined. Mitogen induction of immune interferon was quantitatively associated with mitogen-induced suppressor activity, and cyclic AMP blocked both the suppressor activity and the production of immune interferon in mouse (C57B1/6) spleen cell cultures. The evidence is as follows: (a) The concentrations of dibutyryl cyclic AMP that blocked T-cell mitogen (staphylococcal enterotoxin A) suppressor activity were the same as those that blocked mitogen induction of immune interferon. (b) The blocking action of dibutyryl cAMP on both the suppressor and interferon effects of mitogen was a function of the time of dibutyryl cAMP addition to cultures relative to mitogen addition. (c) A dramatic immunoenhancing effect of mitogen occurred in the presence of dibutyryl cAMP under conditions that blocked production of immune interferon. Specifically, mitogen-induced helper cell function is dramatically enhanced in the presence of dibutyryl cyclic AMP, if the mitogen is added to cultures 24 to 48 hr after SRBC and dibutyryl cyclic AMP. Dibutyryl cyclic GMP did not affect the mitogen- or cyclic AMP-induced effects under the conditions of our test system. Under the conditions described here, then, cyclic AMP appears to selectively block suppressor cell activity while allowing or aiding mitogen-induced helper cell activity. It is possible that the immune response is a reflection of the ratio of helper to suppressor activities in the system.     

Neurochemical and morphological studies on differentiation of NG108-15 cells by phorbol ester and forskolin

12-O-tetradecanoylphorbol 13-acetate (TPA), forskolin or dibutyryl cAMP induced neurite outgrowth and inhibition of cell growth in NG108-15 cells. TPA, forskolin and dibutyryl cAMP significantly increased specific activity of choline acetyltransferase. Forskolin markedly stimulated cAMP accumulation, but not TPA, suggesting that forskolin could induce differentiation by increasing the cAMP content via adenylate cyclase activation, but TPA-induced differentiation seems not to be due to the raise of the cAMP level. Incubation of the cells with TPA, forskolin or dibutyryl cAMP for 24 h resulted in enhancement of 50 mM K⁺-evoked Ca²⁺ influx and neurite elongation, although incubation with these agents for 1 h didn't affect these events. From these results, it is suggested that TPA and forskolin induce differentiation of NG108-15 cells to acetylcholine neurons via different mechanisms: protein kinase C activation by TPA and cAMP-dependent protein kinase activation by forskolin. In addition, it is likely that Ca²⁺ channels in cells differentiated by TPA, forskolin or dibutyryl cAMP become sensitive to depolarization.     

Regulation of immune responses. I. Effects of cyclic AMP and cyclic GMP on immune induction.

Agents that raise intracellular cAMP levels (dibutyryl cyclic AMP, aminophylline, adenosine and butyric acid) increase the magnitude of an in vitro primary humoral immune response when added at 10^?3M during the first 12 hr of a 108 hr culture. Under the same conditions, cGMP has no direct effect but inhibits cAMP-mediated stimulation. DbcAMP (10^?3M or 10^?4M), present from 0 to 12 hr, also increases the number of cytotoxic lymphocytes in CBA/J (H-2^k) spleen cell cultures stimulated in a one-way mixed lymphocyte reaction with DBA/2J (H-2^d) spleen cells. The dbcAMP effect is antigen-dependent in both humoral and cell-mediated immunity and antigen-specific in the case of humoral responses.     

Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells

F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). cDNAs corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl cAMP (Bt₂cAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt₂cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.     

Induction of lysosomal glycosidases by dibutyryl cAMP in neuroblastoma cells

We have studied the regulation of lysosomal glycosidases during morphological differentiation of NB2a neuroblastoma cells. Cells treated with dibutyryl cAMP induced axon-like neurites and showed a 2–4 fold increase in the activity of 6 lysosomal glycosidases, reaching their highest level after 5 days of treatment. Cells treated with retinoic acid, which induced dendrite-like neurites, did not show significant changes in the glycosidases activity although cell proliferation was also inhibited. There was no change in the pattern of the enzyme secretion during the dibutyryl cAMP treatment and morphological analysis using electron microscopy and cytochemical staining with acid phosphatase indicated the presence of lysosomes in the induced neurites.     

Retinoic acid-induced differentiation-specific, C-kinase-dependent phosphorylation of cytosolic 44 and 32 kDa proteins in HL-60 cells

The effect of various differentiation-inducers on the activity of Ca2+, phospholipid-dependent protein kinase (C-kinase) activity and endogenous protein phosphorylation by the kinase were examined in the extracts of HL-60 cells. Although all of the inducers, retinoic acid, dibutyryl cAMP, nicotinamide, dimethylsulfoxide, and 3-aminobenzamide increased the cytosolic C-kinase activity accompanied with the differentiation into mature myelocytes, only retinoic acid markedly enhanced Ca2+, phospholipid-dependent phosphorylation of 44 and 32 kDa proteins in the cytosol. These results suggest that the differentiation pathway induced by retinoic acid is different from the pathways induced by other inducers.     

Plasticity in cultured carotid body chemoreceptors: Environmental modulation of GAP-43 and neurofilament

In this study we use dissociated cell cultures of the rat carotid body to investigate the adaptive capabilities of endogenous oxygen chemoreceptors, following chronic stimulation by various environmental factors. These oxygen chemoreceptors are catecholamine-containing glomus cells, which derive from the neural crest and resemble adrenal medullary chromaffin cells. Using double-label immunofluorescence, we found that chronic exposure of carotid body cultures to hypoxia (2% to 10% oxygen) caused a significant fraction of tyrosine hydroxylase-positive (TH+) glomus cells to acquire detectable immunoreactivity for growth-associated protein gap-43. The effect was dose-dependent and peaked around an oxygen tension of 6%, where approximately 30% of glomus cells were GAP-43 positive. Treatment with agents that elevate intracellular cyclic adenosine monophosphate (cAMP) (i.e., dibutyryl cAMP or forskolin) also markedly stimulated GAP-43 expression. Since hypoxia is known to increase cAMP levels in glomus cells, it is possible that the effect of hypoxia on GAP-43 expression was mediated, at least in part, by a cAMP-dependent pathway. Unlike hypoxia, however, cAMP analogs also stimulated neurofilament (NF 68 or NF 160 kD) expression and neurite outgrowth in glomus cells, and these properties were enhanced by retinoic acid. Nerve growth factor, which promotes neuronal differentiation in related crest-derived endocrine cells, and dibutyryl cGMP were ineffective. Thus, it appears that postnatal glomus cells are plastic and can express neuronal traits in vitro. However, since hypoxia stimulated GAP-43 expression, without promoting neurite outgrowth, it appears that the two processes can be uncoupled. We suggest that stimulation of GAP-43 by hypoxia may be important for other physiological processes, e.g., enhancing neurotransmitter release or sensitization of G-protein–coupled receptor transduction. © 1995 John Wiley & Sons, Inc.     

No relationship between morphology changes and metabolism of α-linolenate and eicosapentaenoate in rainbow trout (Oncorhynchus mykiss) astroglial cells in primary culture

1. Primary cultures of rainbow trout brain astroglial cells, prelabelled with [1-¹⁴C] polyunsaturated fatty acids (PUFA), were treated with various agents and the effects on cell morphology and n-3 PUFA metabolism were investigated.2. The effects of dibutyryl cAMP on trout astroglial cell cultures were similar to those of dibutyryl cAMP on primary cultures of rat brain astroglia, inducing process-bearing morphology.3. Hydrocortisone had the opposite effect to dibutyryl cAMP on the morphology of the trout astroglial cells, reducing the degree of process-bearing morphology.4. Dibutyryl cAMP increased the percentage of [¹⁴C]eicosapentaenoic acid (EPA, 20:5n-3) elongated to 22:5, whereas hydrocortisone increased the percentage of [¹⁴C]linolenic acid (LNA, 18:3n-3) desaturated and elongated to 20:5.5. Peroxisome effectors also affected trout astroglial cell morphology with the peroxisomal proliferator, clofibrate, reducing the degree of process formation, whereas the peroxisomal inhibitor, 3-aminotriazole, increased the degree of process formation.6. However, the peroxisomal effectors had similar effects on n-3 PUFA metabolism, reducing the percentages of [¹⁴C]LNA converted to 20:5 and 22:5, whereas both increased the percentages of [¹⁴C]EPA converted to 22:5 and 22:6.7. We concluded that changes in the morphology of trout astroglial cells in vitro are not directly related to changes in n-3 PUFA metabolism.     

Induction by retinoic acid of NAD+-glycohydrolase activity of myelomonocytic cell lines HL-60, THP-1 and U-937, and fresh human acute promyelocytic leukemia cells in primary culture

HL-60, a human promyelocytic leukemia cell line, is induced to differentiate by retinoic acid to mature granulocytes. We have now found that after the addition of 1 μM retinoic acid to HL-60 cultures an increase in NAD⁺-glycohydrolase (NADase) activity is detected by 6 hr and after a 33-fold increase in activity reaches a plateau by 24 hr. Cycloheximide inhibits completely the retinoic acid-induced increase in NADase activity indicating that enzyme induction requires protein synthesis $\text{de}\text{,}\text{novo}$. An increase of NADase activity was found not only in HL-60 cells but also in two human monoblast cell lines (U-937 and THP-1) and fresh cells in primary culture from two patients with acute promyelocytic leukemia. An increase in synthesis $\text{de}\text{,}\text{novo}$ of NADase does not appear to be obligatory for differentiation of HL-60 because there was no increase of NADase activity in HL-60 cells induced to differentiate with either dimethylsulfoxide, hypoxanthine, butyrate, or 1, 25-dihydroxycholecalciferol and there were marked increases in NADase activity at concentrations of retinoic acid having little or no effect on differentiation.     

Hormonal induction of differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP

It has previously been shown that retinoic acid induces multiple phenotypic changes in cultures of F9 teratocarcinoma stem cells. In this paper we demonstrate that these retinoid-generated cells can be converted to yet another cell type by compounds that elevate cAMP concentrations. The phenotype of the new cell type is characterized by the synthesis of plasminogen activator, laminin and type IV collagen, and by very low levels of alkaline phosphatase and lactate dehydrogenase. The secretion of plasminogen activator and type IV collagen, and low levels of alkaline phosphatase and lactate dehydrogenase, have been previously shown to be properties of parietal endoderm, an extraembryonic cell which is generated early in mouse embryonesis. We show here that parietal endoderm also synthesizes laminin. The cell type generated by retinoic acid and dibutyryl cAMP treatment is therefore indistinguishable from definitive parietal endoderm. Analysis of the final phenotype indicates that it is not dependent upon the continued presence of either compound, and that cAMP agents are active only on cells that have been treated with retinoic acid.     

Pyruvate-Induced changes in hepatoma cell morphology and macromolecular synthesis

Cells maintained in basal growth medium with 0.2–1.0% serum often require citric acid cycle intermediates for optimal viability. We have found that pyruvate added to minimal growth medium causes cellular flattening and formation of external processes accompanied by increaded DNA synthesis in cultured hepatoma cells (HTC cells). Cells were cultured in plalstic T-flasks (0.5, 1.0, or 2.0 × 10⁶ cells/flask) containing 5 ml medium (90% Eagle's Basal Medium (BME) and 10% Swim's S-77) with various concentrations of fetal calf serum (0.2,0.25, 0.5, 1.0, 2.0, 10%) and either pyruvate (50, 100, 250,500, 1,000μg/ml), or one of: dibutyryl cAMP (DBcAMP) or dibutyryl cGMP (DBcGMP) at 10^?3, 10^?4, or 10^?5 M. At 44–48 hr cultures were pulsed with tritiated thymidine, uridine, or lecucine. Cells became attached to the plastic surface within 24hr. Cells in medium with 0.25 to 2.0% serum had a rounded appearance. With added pyruvate, cellular flattening, process formation, and an increased adherence to the substratum was absorbed. By 48 hr, culture without pyruvate grew in rounded clusters; with pyruvate, cells formed extensive interconnecting processes that appeared loosely attached to the monolayer surface. At the cell densities tested, process formation was maximal with 250 to 500 μg/ml pyruvate. Cytochalasin B blocked flattening and process formation; EDTA (1 mg/ml) caused retraction of processes within 3 min, and a slow dissolution of these structures within cells was observed. DBcAMP or DBcGMP did not induce process formation. Flattening and process foormation in pyruvate-enriched cultures were accompanied by marked stimulation of DNA synthesis and smaller increases in RNA and protein synthesis. Cell number was not affected. These pyruvate-induced changes suggest that alterations in energy metabolism, or precursors that enhance viability and macromolecular synthesis in mammalian cell cultures, may exert marked effects on cellular morphology without corresponding changes in growth of neoplastic liver cells.     

Mechanisms of lymphocyte "deletion" by high concentrations of ligand. I. Cyclic AMP levels and cell death in T-lymphocytes exposed to high concentrations of concanavalin A

DNA synthesis, cell survival, and cyclic AMP (cAMP) levels were compared in whole and purified lymph node cells (LNC) cultured with optimal (5 μg/ml) and excess (200 μg/ml) concentrations of native (N) or succinyl (S) concanavalin A (Con A) as possible models for antigen-induced lymphocyte activation and “high-dose” tolerance. Whole LNC cultured with optimal N-Con A or S-Con A showed continuing DNA synthesis and cell viability between 30 and 50% at 48 hr. In contrast, with excess N-Con A, they showed virtually no [³H]TdR uptake at this time and there was progressive loss of cell viability beginning at 8 hr; by 48 hr almost no viable cells remained. Excess S-Con A induced little cell death up to 24 hr, but by 48 hr only 20% of the cells initially placed in culture remained alive and sythesized DNA. Intracellular cAMP showed a transient rise in cultures stimulated with optimal N-Con A, peaking at 15 min, then returning to normal levels, to rise again between 24 and 48 hr. With excess N-Con A, cAMP rose within 15 min and continued to increase to a peak at 24 hr. cAMP levels in the presence of excess S-Con A remained at control levels for the first 24 hr and increased between 24 and 48 hr. LNC depleted of macrophages and B cells, when cultured in excess N-Con A, had an inhibition of DNA synthesis, elevated cAMP levels, and cell death comparable to whole LNC. It seems unlikely, however, that the increase in cAMP mediates cell killing since cAMP was not elevated yet cell death occurred in nylon wool-purified T cells exposed to excess N-Con A. Dibutyryl cAMP, and prostaglandin E₁, which markedly increase cAMP levels, failed to kill LNC at doses which totally inhibited DNA synthesis, and cells of the mouse T-lymphoma S49 and its cAMP-dependent protein kinase-deficient variant were killed equally by excess N-Con A. It is suggested that a sustained elevation of either cAMP or Ca²⁺ after early commitment may provide a significant mechanism of tolerogenesis.     

Inhibition by islet-activating protein, pertussis toxin, of retinoic acid-induced differentiation of human leukemic (HL-60) cells

Human promyelocytic leukemic (HL-60) cells were induced to differentiate into neutrophil- or macrophage-like cells by incubation of the cells with retinoic acid, dibutyryl cyclic AMP (Bt2cAMP) or phorbol 12-myristate 13-acetate (PMA). Differentiation was determined by an increase in the percentage of morphologically mature cells. The retinoic acid-induced differentiation of HL-60 cells was, but the Bt2cAMP- or PMA-induced one was not, inhibited by prior exposure of the cells to islet-activating protein (IAP), pertussis toxin. The IAP-induced inhibition was correlated with the toxin-catalyzed ADP-ribosylation of a membrane GTP-binding protein with a molecular mass of 40 kDa. Thus, the IAP-substrate GTP-binding protein appears to be involved in the retinoic acid-induced differentiation of HL-60 cells.     

Studies on the effect of retinoids on the differentiation of teratocarcinoma stem cells in vitro and in vivo

Previous work has shown that retinoic acid can induce F9 embryonal carcinoma cells to differentiate into extraembryonic endoderm in vitro. We have investigated in detail the structureactivity relationship of various derivatives of retinoic acid, the kinetics of the cellular response, and the nature of the type of endoderm formed. These experiments demonstrate that the carboxylic acid group of retinoic acid is essential for activity, whereas the structure can be modified in the cyclohexenyl ring system with retention of effectiveness. The time course of the response of the cells shows that the amount of differentiation is proportional to the duration of treatment, and suggests that the retinoid facilitates formation of endoderm at each division. Finally, even well-differentiated cultures do not produce alpha-fetoprotein, indicating that the endoderm formed is similar to parietal, but not visceral, endoderm. We have also explored the consequence of in vivo retinoic acid treatment on the tumorigenesis of F9 embryonal carcinoma cells in 129/Sv mice. The presence of retinoic acid in the diet at 100 mg/kg significantly retarded tumor growth and lengthened the survival time of the mice. Histological analysis and assays for biochemical markers of endoderm in the tumor both suggest that the retinoid is inducing differentiation of the embryonal carcinoma cells in vivo.