Effects of dibutyryl cAMP and bromodeoxyuridine on expression ofN-acetylglucosaminyltransferases III and V in GOTO neuroblastoma cells

The sugar chain structures of the cell surface change dramatically during cellular differentiation. A human neuroblastoma cell line, GOTO, is known to differentiate into neuronal cells and Schwannian cell-like cells on treatments with dibutyryl cAMP and bromodeoxyuridine, respectively. We have examined the expression of UDP-N-acetylglucosamine: -d-mannoside -1,4N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) and UDP-N-acetylglucosamine: -6-d-mannoside -1,6N-acetylglucosaminyltransferase V (GnT-V: EC 2.4.1.155), two major branch forming enzymes inN-glycan synthesis, in GOTO cells on two distinct directions of differentiation.In neuronal cell differentiation, GnT-III activity showed a slight increase during initial treatment with Bt₂cAMP for 4 days and decreased drastically after the fourth day, but the mRNA level of GnT-III did not show a decrease but in fact a slight increase. GnT-V activity increased to approximately two- to three-fold the initial level with increasing mRNA level after 8 days, and lectin blot analysis showed an increase in reactivity toDatsura stramonium (DSA) of the immunoprecipitated neural cell adhesion molecule (NCAM). In Schwannian cell differentiation, the activity and mRNA level of GnT-III showed no significant change on treatment with BrdU. GnT-V activity also showed no change in spite of the gradual increase in the mRNA level. These results suggest that the activation of GnT-V during neuronal cell differentiation of GOTO cells might be a specific change for branch formation in N-glycans, and this affects the sugar chain structures of some glycoproteins such as NCAM.Abbreviations and trivial names GnT N-acetylglucosaminyltransferase - Bt₂cAMP N ⁶,O ⁶-dibutyryl cAMP - BrdU bromodeoxyuridine - DSA Datsura stramonium - NCAM neural cell adhesion molecule - PAGE polyacrylamide gel electrophoresis     

Myogenesis in vitro. Enhancement by dibutyryl cAMP

Cholera enterotoxin (CT) increased the concentration of adenosine 3′-5′-cyclic monophosphate (cAMP) in monolayer cultures of adrenal tumor cells after a 60 min lag phase in contrast to the rapid effect of adrenocorticotropin (ACTH). The change in intracellular cAMP was accompanied by the release of steroids into the culture medium and a reversible alteration of monolayer morphology.     

Induction of differentiation in mouse neuroblastoma cells by hexamethylene bisacetamide

Hexamethylene bisacetamide (HMBA), a potent inducer of erythroid differentiation in murine erythroleukemia cells (1), induces differentiation in mouse neuroblastoma cells, as indicated by the extension of neurites and the development of an excitable membrane. HMBA is effective at concentrations 50-fold lower than dimethylsulfoxide (2), another inducer of differentiation in both mouse neuroblastoma and murine erythroleukemia cells.     

Induction of reverse transformation and normal cell cycle regulation by dibutyryl cAMP in a chemically transformed cell line

The objective of this study was to determine whether N6, O2-dibutyryl 3',5'-adenosine monophosphate (db-cAMP)-induced reverse transformation in a chemically transformed mouse cell line, AKR-MCA, would restore normal cell cycle regulation, particularly with regard to their growth arrest in the early G1 period. The AKR-MCA cells were grown to confluency in the presence or absence of db-cAMP (0.5 mM) plus theophylline (1 mM). The confluent cultures were trypsinized and a portion of the cells were fused with mitotic HeLa cells to induce premature chromosome condensation, while the remaining cells were used to study the kinetics of initiation of DNA synthesis. The prematurely condensed chromosomes (PCC) of the control and the treated cultures were classified into G1, S, or G2 types on the basis of their morphology. The G1 PCC were further subclassified into six groups (+1 - +6); +1 being the most condensed and +6 the most decondensed. The cyclic AMP (cAMP)-treated cells exhibited better attachment to the culture dish, were blocked in early G1 period at confluency, and entered S phase about 4 h later than the control following subculturing. In contrast, a majority of cells in the control cultures were arrested in S phase at confluency. These data indicate that the db-cAMP-induced reverse transformation in AKR-MCA cells at least partially restores normal cell cycle regulation in these chemically transformed cells.     

Specific inactivation of lysosomal glycosidases in living fibroblasts by the corresponding glycosylmethyl-p-nitrophenyltriazenes.

Glicentin (a highly purified 100-amino acid peptide with glucagon-like immunoreactivity from porcine gut) was subjected to limited digestion with trypsin and carboxypeptidase B, and the resulting peptides were studied by gel filtration and region-specific glucagon radioimmunoassays. Similar digests of glucagon and purified fragments of glucagon were studied in parallel. Glicentin gave rise to peptides that corresponded closely to the 1-17 and 19-29 fragments of glucagon. Also, 125I-labelled glicentin and 125I-labelled glucagon gave rise to identical fragments after trypsin treatment. On the basis of this and other evidence [Jacobsen, Demandt, Moody & Sundby (1977) Biochim. Biophys. Acta 493, 452-459] it is concluded that glicentin contains the entire glucagon sequence at residues number 64-92 and thus fulfills one of the requirements for being a 'proglucagon'.     

Arrest of cultured rat liver cells in G2 phase by the treatment with dibutyryl cAMP.

When rat liver cells which had been grown in a protein- and lipid-free synthetic medium were incubated in the presence of dibutyryl adenosine 3′:5′-cyclic monophosphate (But₂cAMP) and theophylline, labeling index of cells with [³H]-thymidine was decreased. Percentage of cells which had twice the amount of DNA per nucleus compared to the basic value increased during the cultivation of cells in the presence of the drugs. Mitotic index increased immediately after the removal of the drugs. From these results, it was concluded that But₂cAMP arrested the cell cycle mainly at G2 phase.     

Muscarinic receptor-mediated increase in cAMP levels in SK-N-SH human neuroblastoma cells

Stimulation of muscarinic cholinergic receptors in SK-N-SH human neuroblastoma cells resulted in a 1.5-4 fold increase in intracellular cAMP levels. This unusual response was sensitive to atropine and pirenzepine but insensitive to pertussis toxin. It was observable regardless of whether basal, PGE1- or forskolin-stimulated cAMP levels were measured. The half-maximal concentration for carbachol-stimulation of cAMP levels (6 microM) was similar to that for the previously determined carbachol-induced stimulation of phosphoinositide turnover in these cells, suggesting that the former is mediated by the latter. These data indicate that cross-talk between the phosphoinositide turnover system and the adenylate cyclase system results in increased cAMP levels in SK-N-SH cells in response to muscarinic receptor stimulation.     

Induction of axon-like and dendrite-like processes in neuroblastoma cells

Neuroblastoma cells are widely utilized models for the study of the neuritic outgrowth phase of neuronal differentiation, but relatively few such studies have attempted to identify the nature of the process outgrowths. This identification will be necessary in developing strategies for utilizing these models to distinguish the underlying mechanisms involved in axonogenesis vs dendritogenesis. In an effort to identify procedures for inducing specific types of neurite outgrowth, and for distinguishing axon- from dendrite-like processes, we have subjected two neuroblastoma cell lines to a variety of stimuli previously shown to induce neurite outgrowth in these cells. These include neuraminidase, ionomycin, KCl+dibutyryl cAMP, cholera toxin B subunit, retinoic acid, dibutyryl cAMP (alone), GM1 ganglioside, and low serum. The first four of these (group 1) gave rise to neurites with axon-like characteristics, including immunostaining that was positive for phosphorylated high molecular weight neurofilament protein (NF-H) and synaptic vesicle protein-2 (SV2), but negative for microtubule-associated protein-2 (MAP2). The next three treatments (group 2) resulted in dendrite-like processes, as evidenced in immunostaining that was positive for MAP2 and negative for NF-H and SV2. Neurites produced by low serum had mixed properties. These cytoskeletal differences were supported by immunoblot analysis with antisera to the above cytoskeletal proteins. Striking morphological differences were also noted, group 2-induced neurites being significantly shorter with more branch points than those generated by group 1 stimulants. Time of exposure to stimulatory agent was crucial in determining expression of the neuritic phenotype. Correlation with previous studies suggests that axon-like neurites result from stimulants which elevate intracellular Ca2+, a dependence not previously reported to our knowledge. Dendrite-like process outgrowth, on the other hand, does not appear to depend on altered intracellular Ca2+.     

Effect of dibutyryl cAMP on cell cycle progression of rat brain tumor cells in vitro

Summary Autoradiographic and flow microfluorometry analyses have been applied to a study of perturbed cell kinetics in 9L rat brain tumor cells treated with dibutyryl cyclic AMP and theophylline alone and in combination in vitro. At a concentration of 1 mM each, cell growth ceased shortly after the administration of these drugs. The results indicate that cells in S and G₂ phase at the time of drug administration can undergo mitosis even though a considerable prolongation of G₂ phase was apparent. However, cells in G₁ at the time of drug administration were arrested in that phase whereas those cells in S or G₂ were able to complete one mitosis before becoming arrested in the G₁ phase. This blocking effect was reversible, and cells resumed proliferation at a normal rate shortly after the removal of these drugs. This work was supported in part by NIH Cancer Research Center Grant CA-13525 and CA-19992 from NCI, and by the Association for Brain Tumor Research. Presented at the 6th International Cell Cycle Conference, March, 1976, New Orleans, Louisiana. The tumor used in this study was provided by William H. Sweet, Paul T. Kornblith, Janette L. Messer and Beverly O. Whitman of the Massachusetts General Hospital, Boston, Massachusetts.     

Activation of adrenal sterol ester hydrolase by dibutyryl cAMP and protein kinase

Direct activation of adrenal Sterol Ester Hydrolase (EC 1.1.1.13) by dibutyryl cAMP, ATP and Mg⁺² has been demonstrated in adrenal homogenates from three species. Variability in the degree of activation was minimized by preincubation of the tissue homogenate for two hours prior to addition of the cofactors and subsequent enzyme assay. Although baseline sterol ester hydrolytic activity, independent of cofactors, was present in all subcellular fractions, the cAMP-dependent enzyme was primarily associated with the 105,000 × g soluble fraction of the cell. A requirement for protein kinase in the system was demonstrated with a partially-purified enzyme from bovine adrenal.     

Effect of dibutyryl cAMP on cell cycle progression of rat brain tumor cells in vitro.

Autoradiographic and flow microfluorometry analyses have been applied to a study of perturbed cell kinetics in 9L rat brain tumor cells treated with dibutyryl cyclic AMP and theophylline alone and in combination in vitro. At a concentration of 1 mM each, cell growth ceased shortly after the administration of these drugs. The results indicate that cells in S and G2 phase at the time of drug administration can undergo mitosis even though a considerable prolongation of G2 phase was apparent. However, cells in G1 at the time of drug administration was arrested in that phase, whereas those cells in S or G2 were able to complete one mitosis before becoming arrested in the G1 phase. This blocking effect was reversible, and cells resumed proliferation at a normal rate shortly after the removal of these drugs.     

Induction of lysosomal enzymes in contact inhibited 3T3 cells

The pattern of changing activities of three lysosomal enzymes, N-acetyl-β-D-glucosaminidase, aryl sulfatase, and DNase II, and that of DNA polymerase was followed in homogenates of 3T3 cells during the logarithmic phase of growth and in stationary cultures. The change in activities of the polymerase and the lysosomal enzymes is antiparallel. DNA polymerase exhibits highest activity in growing cultures, and shows a three-fold decline of the specific activity in stationary cultures. The lysosomal enzymes show a very marked increase in their specific activity after density saturation is reached, which can be prevented by the addition of cycloheximide. Colcemid added to logarithmically growing cultures also causes an increase in the specific activities of lysosomal enzymes.     

A TSH/dibutyryl cAMP activated Cl-/I- channel in FRTL-5 cells.

An iodide (I) and chloride (Cl) channel has been identified in the continuously cultured FRTL-5 thyroid cell line using a cell attached patch clamp technique. The channel is activated by TSH and dibutyryladenosine cyclic monophosphate (Bt2-cAMP) but not by phorbol 12-myristate 13-acetate (TPA). Gluconate can not replace chloride or iodide and the channel is impermeable to Na+,K+ and tetraethylammonium ions. The current-voltage relationship demonstrates that the single channel current is a linear function of the clamp voltage. Single channel currents reversed at a pipette potential close to 0 mV. The mean single channel conductance was 60 pS for Cl- and 50 pS for I-. From the I-V relationship there was a strong outward rectification with Cl-, and a complete block with I-, in the single channel current above +40 mV. The feature of the channel is manifested in the single channel records by four distinct, equally spaced conductance levels. We suggest the channel is important for the transport of I and Cl ions across the apical membrane into the colloid space and is important for hormone synthesis and follicle formation.     

Induction of axonal differentiation by silencing plasma membrane-associated sialidase Neu3 in neuroblastoma cells

A reduction of 70% of the plasma membrane-associated sialidase Neu3 activity, due to a corresponding reduction of the enzyme expression by transducing cells with a short hairpin RNA encoding a sequence target (complementary messenger of mouse Neu3), caused neurite elongation in Neuro2a murine neuroblastoma cells. The differentiation process was accompanied in parallel by an increase of the acetylcholinesterase activity, a moderate increase of the c-Src expression and by the presence of the axonal marker tau protein on the neurites. The sphingolipid pattern and turnover in transduced and control cells were characterized by thin layer chromatography, mass spectrometry and metabolic radiolabeling after feeding cells with tritiated sphingosine. Control cells contained about 2 nmol of gangliosides/mg cell protein. GM2 was the main compound, followed by GD1a, GM3 and GM1. In Neu3 silenced cells, the total ganglioside content remained quite similar, but GM2 increased by 54%, GM3 remain constant, and GM1 and GD1a decreased by 66% and 50%, respectively. Within the organic phase sphingolipids, ceramide decreased by 50%, whereas the sphingomyelin content did not change in Neu3 silenced cells.     

Purification and immunological quantification of rat liver lysosomal glycosidases.

Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.