Modification of response of mouse neuroblastoma cells in culture by serum type

Summary The effect of various types of serum on morphological and biochemical changes in mouse neuroblastoma cells (clone NBP₂) in culture was studied. The extent of spontaneous morphological differentiation varied markedly depending upon the type of serum and was maximal in agammaglobulin calf serum (CS). The extent of morphological differentiation after treatment of cells with cyclic AMP-stimulating agents was also dependent upon serum type and was least pronounced in fetal calf serum. The doubling time and extent of clumping varied with the type, of serum. The activity of tyrosine hydroxylase (TH) in NB cells was dependent upon serum type and it was highest in newborn CS and agammaglobulin CS. Although elevation of intracellular levels of cyclic AMP in NBP₂ clone invariably stimulates neurite formation and TH activity, these functions were increased in certain sera without a significant increase in the cellular cyclic AMP levels. The present study shows that neurite formation, growth rate and TH activity are regulated by more than one mode, one of which is mediated by cyclic AMP. The above changes are independently regulated in the sense that the expression of one can be increased in the absence of others. Preliminary reports of this work were presented at the Symposium on Cell Differentiation and Neoplasia, March 1976; American Society for Neurochemistry, March 1978; and the FASEB meetings, April 1978. This work was supported in part by NIH Grant ROESNS 01576, NIH Training Grant 4007072 and Research Scientist Career Development Award MH 42479.     

CYCLIC GMP IN A NEUROBLASTOMA CLONE: POSSIBLE INVOLVEMENT IN MORPHOLOGICAL DIFFERENTIATION INDUCED BY DIBUTYRYL CYCLIC AMP

Abstract— DBcAMP induces morphological differentiation of mouse neuroblastoma cells grown in culture. DBcGMP or 8-Br cyclic GMP when added alone also induces a discrete morphological differentiation. When analogues of cyclic GMP were added together with dBcAMP, neurite outgrowth was strikingly enhanced as compared to the effect of dBcAMP alone. Intracellular concentrations of cyclic GMP were increased during dBcAMP treatment and cyclic AMP levels were increased during 8-Br cyclic GMP treatment. Both treatments produced an increased protein kinase activity, supporting the possibility that not only cyclic AMP but also cyclic GMP may be involved in the differentiation process.     

Multiple Neurite Formation in Neuroblastoma Cell Lines by Griseolic Acid, a Potent Inhibitor of Cyclic Nucleotide Phosphodiesterases

The morphological change of several neuroblastoma cell lines induced by griseolic acid, a novel and potent inhibitor of cyclic nucleotide phosphodiesterase (PDE), was examined. In the cell lines tested, Neuro-2a (a murine neuroblastoma cell line) showed dose-dependent (1 microM-1 mM) neurite extension. Griseolic acid markedly increased the intracellular cyclic AMP level of Neuro-2a cells, suppressed DNA synthesis (82% at 1 mM), and induced multipolar (multiple-neurite-bearing)-type neuritogenesis. A similar type of neurite outgrowth was induced by 8-bromo-cyclic AMP, which also elevated the intracellular cyclic AMP concentration. In contrast, when Neuro-2a cells were treated with retinoic acid, neurite formation was of the monopolar (single-neurite-bearing) type. Papaverine and theophylline, which have been frequently used as PDE inhibitors, failed to induce these morphological changes up to 1 mM, probably owing to the lesser potency of these compounds as compared with griseolic acid on the inhibition of PDE. Retinoic acid, theophylline, and papaverine were ineffective at elevating the intracellular cyclic AMP level. These results suggest that multipolar-type cell shape change in Neuro-2a cells is correlated with the accumulation of intracellular cyclic AMP and that griseolic acid is a useful compound to induce neuroblastoma cells into terminal differentiation.     

Induction of osteoblastic cell differentiation by forskolin. Stimulation of cyclic AMP production and alkaline phosphatase activity

The effects of forskolin on differentiation of osteoblastic cells (clone MC3T3-E1) cultured in alpha-minimum essential medium containing 0.1% bovine serum albumin were investigated by assays of intracellular cyclic AMP level and alkaline phosphatase activity in the cells. Forskolin increased cyclic AMP production in the cells in a dose-related manner, the maximum increase being 250-fold above that of the controls. Alkaline phosphatase activity in the cells was also elevated as early as 24 h and rose to nearly its maximum at 48 h. The elevation was dose-dependent, with a maximum increase at 5 X 10(-6) M forskolin. Forskolin and prostaglandin E2 showed a supraadditive effect on cyclic AMP production in the cells and had an additive effect on alkaline phosphatase activity, whereas forskolin and dibutyryl cyclic AMP had little additive effect on either cyclic AMP production or enzyme activity. These results suggest that cyclic AMP is closely linked to the differentiation of osteoblastic cells in vivo.     

Glucocorticoids Increase Tyrosine Hydroxylase Activity in Cultured Murine Neuroblastoma

Cyclic AMP and glucocorticoids appear to have a role in regulating the activity of tyrosine hydroxylase (TH), as well as the expression of "morphological differentiation" in murine neuroblastoma. Monolayer cultures of C-1300 murine neuroblastoma (clone NBP2) were treated with the following compounds in ethanol: dexamethasone, triamcinolone acetonide, hydrocortisone, cortexolone, androstenedione, testosterone, estradiol-17 beta; or with the phosphodiesterase inhibitor Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone]. Treatment with either 200 micrograms/ml Ro20-1724 or 50 micrograms/ml dexamethasone produced significant increases in TH activity compared to alcohol controls (1.44 vs. 0.82 nmol 14CO2/mg protein/hr compared to 0.095). Triamcinolone acetonide or hydrocortisone also produced smaller, but significant, increases in TH activity compared to dexamethasone. When steroid activities were compared at 25 microM concentration and after 60 min of incubation (to maximize TH activity), triamcinolone acetonide was not as effective (62%) as dexamethasone. The relatively inactive glucocorticoid cortexolone produced a slight but significant increase, while the androgens androstenedione and testosterone and the estrogen estradiol-17 beta were without effect.     

Two Types of Apoptotic Cell Death of Rat Central Nervous System-Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 m M DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.     

Neurite development in vitro. I. The effects of adenosine 3′5′-cyclic monophosphate (cyclic AMP)

Elevated levels of 3′5′ adenosine monophosphate (cyclic AMP) stimulate a wide variety of cellular events including aggregation, differentiation, morphological expression, pigment migration, and secretion. The role of cyclic AMP in these events prompted our present study of embryonic chick dorsal root ganglia. Test substances were applied to cultures during the routine feeding procedure. Their development was quantitatively evaluated on the basis of explant size, length of glial-like outgrowth, distribution of growth, neurite number, length, diameter, and degree of arborization. These parameters were all shown to be independent of each other. The high variability of in vitro neurite development necessitated the use of over 100 cultures per treatment group. Cultures treated with 5′ AMP exhibited no significant differences from controls. Those treated with cyclic AMP, dibutyryl cyclic AMP, or Nerve Growth Factor (NGF) exhibited statistically significant increases in area of outgrowth, the number of neurites per culture, and in diameters, lengths, and degree of neurite arborization. The growth promoting activity of dibutyryl cyclic AMP and NGF were greater than those of cyclic AMP. Electron microscopic study shows neurites formed under the influence of cyclic AMP or its dibutyryl derivative to resemble those grown in NGF. These studies suggest the possibility that cyclic AMP stimulates neurite growth by mediating the process of microtubule (MT) assembly. They further prompt us to speculate that one way NGF enhances neurite development is by stimulating MT assembly via a “Second Messenger System”.     

Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts.

Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.     

Serum modification of cyclic nucleotide phosphodiesterase forms independent of protein synthesis.

Addition of 10% fetal calf serum to BHK cells made quiescent by maintenance for 48 hours in sub-optimal serum (0.5%) caused rapid changes in cyclic AMP phosphodiesterase activity (increased maximum velocity and affinity) even in the presence of inhibitors of protein synthesis. Activity changes were associated with an alteration in the number of forms of cyclic AMP phosphodiesterase identified by Agarose gel filtration. Three forms of cyclic nucleotide phosphodiesterase were apparent after serum addition whereas only two forms were resolved in quiescent BHK cells. The initial rapid increase in cyclic AMP phosphodiesterase activity seen when serum was added to quiescent cells was followed temporally by a much slower increase in cyclic AMP phosphodiesterase activity that could be prevented by cycloheximide or actinomycin D.     

Effects of adenosine 3'':5''-cyclic monophosphate and serum on synthesis of hyaluronic acid in confluent rat fibroblasts.

A small amount of hyaluronic acid is synthesized in confluent cultures of rat fibroblasts, which have a high content of cyclic AMP. Addition of calf serum caused a rapid decrease in the cellular cyclic AMP content and large increases in hyaluronic acid synthetase activity and hyaluronic acid production. Addition of cyclic AMP also caused a marked increase in hyaluronic acid synthetase activity within 2h and then increased hyaluronic acid production. The effects of cyclic AMP and serum on hyaluronic acid synthesis were additive. Prostaglandin E2, which increased the cyclic AMP by stimulating adenylate cyclase, was as effective as cyclic AMP in increasing hyaluronic acid synthetase activity, but AMP was far less effective than cyclic AMP. These results indicate that cyclic AMP itself stimulates the mucopolysaccharide synthesis and that the effect of serum is not due to a decrease in cyclic AMP in the cells.     

Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP.

A mutant clone resistant to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP-dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expressed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and express two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.     

Cholera Toxin and Dibutyryl Cyclic AMP Inhibit the Expression of Neurofilament Protein Induced by Nerve Growth Factor in Cultures of Naive and Primed PC12 Cells

The effects of nerve growth factor (NGF), dibutyryl cyclic AMP (db cAMP), and cholera toxin on neurofilament protein expression in cultures of PC12 rat pheochromocytoma cells were examined using an enzyme-linked immunoadsorbent assay (ELISA). Morphological differentiation induced by NGF was associated with up to 30-fold increases in the level of neurofilament protein recognised by monoclonal antibody RT97. A more rapid response was apparent from primed as compared to naive PC12 cells. Cholera toxin and db cAMP both induced morphological differentiation of naive PC12 cells, but failed to promote neurite regeneration from primed cells. Neither response was associated with a significant induction of neurofilament protein. Both cholera toxin and db cAMP, but not B-cholera toxin nor antibodies to the toxin receptor, were found to inhibit the neurofilament protein response induced by NGF. Primed cells were more susceptible to this inhibition, and both cholera toxin and db cAMP inhibited neurite regeneration from these cells. These data suggest that increased intracellular cyclic AMP can suppress the expression of neuronal differentiation antigens induced by NGF, and are consistent with a role for neurofilament protein in promoting or facilitating the formation of a stable neuritic network.     

N<Superscript>6</Superscript>,O<Superscript>2</Superscript>′-Dibutyryl Adenosine 3′,5′-Monophosphate induces Pigment Production in Melanoma Cells

IN VITRO studies have suggested that adenosine 3′,′-monophosphate (cyclic AMP) regulates cell morphology. During treatment with the dibutyryl analogue of cyclic AMP, N⁶,O²′-dibutyryl cyclic AMP, transformed fibroblasts acquire several morphological characteristics of untransformed fibroblasts^1,3. Cell processes are extended, the cells occupy a greater surface area and in some cases there is a parallel alignment of cells. Chinese hamster ovary cells are affected in the same way. In neuroblastoma cells⁵, dibutyryl cyclic AMP induces neurite extension and increases the activity of acetylcholinesterase, an indicator of biochemical differentiation⁶. Cyclic AMP is known to control the dispersion of melanin^7,8 and the differentiation of melanoblasts into melanocytes. We have now found that during treatment with dibutyryl cyclic AMP, melanoma cells spread out, appear larger and produce considerably more pigment than untreated cells.     

STIMULATORY EFFECTS OF SUBSTANCE P ON NEURITE EXTENSION AND CYCLIC AMP LEVELS IN CULTURED NEUROBLASTOMA CELLS

Abstract— Synthetic substance P initially increased cyclic AMP levels and subsequently induced neurite extension in cultured neuroblastoma N 18 cells. The magnitude of these effects depended on the concentration of fetal calf serum (FCS) in the culture medium, being more evident in the presence of a lower (0.1%) concentration of FCS.
In Eagle's medium containing 0.1% FCS, low concentrations of substance P (10⁻⁷-10⁻⁵ M) increased cyclic AMP levels and stimulated neurite extension.
In Eagle's medium containing 5%FCS, both substance P at concentrations of 10⁻⁵-10⁻³M and dibutyryl cyclic AMP at concentrations of 10⁻⁴-10⁻²M increased cyclic AMP levels and stimulated neurite extension. The activities of acetylcholinesterase, (Na⁺+ K⁺)-, HCO₃⁻ and Mg²⁺ -stimulated-ATPase were also increased. Cell growth was inhibited.
Substance P at concentrations of 10-⁷-10⁻⁵M also stimulated the adenylate cyclase activity of a particulate fraction of N 18 in a concentration-dependent manner.     

Effects of sera types and cell density on the concentration of cyclic nucleotides in normal and simian virus transformed mouse fibroblasts

The effects of serum and cell density on the concentration of cyclic AMP, cyclic GMP in normal mouse fibroblasts cells (3T3 cells) and their Simian Virus 40 transformed derivative (SV3T3 cells) were studied. 3T3 cells grown in 10% foetal bovine serum exhibit density dependent inhibition of growth and associated with this in an increase in the concentration of cyclic AMP, a decrease in the concentration of cyclic GMP and an increase in the ratio (cyclic AMP/cyclic GMP) of the cyclic nucleotides. 3T3 cells grown in 10% newborn calf serum exhibit a higher saturation density and this is associated with a low concentration of cyclic AMP and a high concentration of cyclic GMP. SV3T3 cells grown in either 10% foetal bovine serum or 10% newborn calf serum show high saturation densities and this is associated with a low and decreasing concentration of cyclic AMP and a high concentration of cyclic GMP. When the level of the cyclic AMP in both cell lines was artificially raised by adding dibutyryl cyclic AMP and theophylline to the growth media, the cells grew to low densities.     

Differential effect of sodium butyrate on cyclic AMP phosphodiesterase activities in butyrate sensitive and resistant mastocytoma cells

The effect of sodium butyrate on the intracellular cyclic AMP levels and the activities of cyclic AMP-regulating enzymes were examined in two types of mastocytoma p-815 cells in culture: one type (S cell) was sensitive and the other (R cell) was resistant to the induction of differentiation by sodium butyrate. In the presence of sodium butyrate, adenylate cyclase activity increased in both S and R cells to the same degree, whereas the level of cyclic AMP was elevated only in S cells. Cyclic AMP phosphodiesterase activity increased in R cells but not in S cells. Cyclic AMP phosphodiesterase activities of two cell populations differed in their response to sodium butyrate and they seem to have an important role in regulating cellular level of cyclic AMP that might be an important factor in controlling cell differentiation.     

Extracellular Calcium-Induced Neuroblastoma Cell Differentiation: Involvement of Phosphatidylinositol Turnover

The rat CNS neuroblastoma B50 cell line is known to differentiate on addition of 1 mM dibutyryl cyclic AMP or on withdrawal of serum. In this report it is shown that high levels of extracellular calcium (10-25 mM) cause neurite extension, an important component of morphological differentiation. Stimulation of calcium influx with the ionophore A 23187 or blockade of calcium efflux with lanthanum are less efficient than extracellular calcium in stimulating neurite extension. These data suggest that intracellular calcium is not sufficient to cause full expression of a calcium-dependent differentiated state. Furthermore, phosphatidylinositol turnover is sharply altered as early as 1 h after addition of calcium to the medium while cyclic nucleotide levels remain unaffected. This suggests that activation of the phosphatidylinositol second-messenger system by calcium at the level of the cell membrane is the initial step in the cascade of events leading to neurite extension. Later events include a decrease in DNA synthesis (6-10 h after addition of calcium), and increase in intracellular calcium levels (12-24 h after calcium addition) concurrent with neurite extension. The intracellular increase in calcium levels is facilitated by synergistic action of 1 mM dibutyryl cyclic AMP with high external calcium (10-25 mM). This combined treatment results in a more complex pattern of neurite formation characterized by many synaptic-like junctions; this pattern is not obtained when either dibutyryl cyclic AMP or calcium is used as sole inducer.     

Neuronal differentiation of mouse neural crest cells in vitro

The purpose of the present study is to analyze the effect of serum or chick embryo extract (CEE) on the neuronal differentiation of the mouse neural crest cells. When the crest cells were cultured in the medium containing serum at low concentration (5% calf serum), neurite outgrowth was observed. The active outgrowth was detected at 3-4 days in culture. However, in the medium supplemented with 20% calf serum, no neurite appeared, and the crest cells remained fibroblast-like. The differentiation of adrenergic neurons was observed when the crest cells were cultured in the medium containing CEE along with serum.     

The guanine nucleotide exchange factor, C3G regulates differentiation and survival of human neuroblastoma cells

Neuronal differentiation involving neurite growth is dependent on environmental cues which are relayed by signalling pathways to actin cytoskeletal remodelling. C3G, the exchange factor for Rap1, functions in pathways leading to actin reorganization and filopodia formation, processes required during neurite growth. In the present study, we have analyzed the function of C3G, in regulating neuronal cell survival and plasticity. Human neuroblastoma cells, IMR-32 induced to differentiate by serum starvation or by treatment with nerve growth factor (NGF) or forskolin showed enhanced C3G protein levels. Transient over-expression of C3G stimulated neurite growth and also increased responsiveness to NGF and serum deprivation induced differentiation. C3G-induced neurite growth was dependent on both its catalytic and N-terminal regulatory domains, and on the functions of Cdc42 and Rap1. Knockdown of C3G using small hairpin RNA inhibited forskolin and NGF-induced morphological differentiation of IMR-32 cells. Forskolin-induced differentiation was dependent on catalytic activity of C3G. Forskolin and NGF treatment resulted in phosphorylation of C3G at Tyr504 predominantly in the Golgi. C3G expression induced the cell cycle inhibitor p21 and C3G knockdown enhanced cell death in response to serum starvation. These findings demonstrate a novel function for C3G in regulating survival and differentiation of human neuroblastoma cells.