Expression of external-surface membrane proteins in differentiated and undifferentiated mouse neuroblastoma cells.

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N-[3H]-trimethylamino-beta-alanyl-N-hydroxy-succinimide ester ([3H]Me3N-beta Ala-NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiated state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal-specific, while others were common to several clones. Two proteins of Mr 55,000 and 65,000 were labeled by both 125I/lactoperoxidase and Me3N-[3H]-beta Ala-NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150,000 and 200,000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95,000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethyl-sulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.     

Differences in plasma membrane organization of neuroblastoma cells grown in the differentiated and undifferentiated states.

Rabbit skeletal muscle creatine kinase is inactivated when stored at ?17 °C in the presence of either chloride or nitrate. Other anions are not effective. Associated with the inactivation is an altered electrophoretic mobility and the loss of four out of the eight titratable thiol groups in the dimeric catalytic protein of molecular weight 82,600. The altered inactive form is separated from the native active enzyme by electrofocusing, and its catalytic activity is restored by treatment with 2-mercaptoethanol. Gel electrophoresis in the presence and absence of 2-mercaptoethanol establishes that solutions of the inactive enzyme are heterogeneous, containing mostly a protomeric polypeptide of molecular weight 41,000, but also significant amounts of disulfide-linked dimers, trimers, and tetramers. Sedimentation equilibrium analysis confirms the existence of higher molecular weight aggregates along with the preponderant protein species of molecular weight 43,000.     

Difference in putrescine transport in undifferentiated versus differentiated mouse NB-15 neuroblastoma cells

$\text{Clostridium}\text{histolyticum}$ collagenase has been chemically modified with a series of reagents to identify essential amino acid residues. The activity of the enzyme is not significantly altered by the seryl reagents diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, the cysteinyl reagents p-chloromercuribenzoate and iodoacetamide, or the arginyl reagents butanedione and phenylglyoxal. The enzyme is inactivated by 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide and N-ethyl-5-phenylisoxazolium-3′-sulfonate, indicating the presence of an essential carboxyl residue. Both acetylimidazole and tetranitromethane inactivate the enzyme and the acetylimidazole reaction is reversed by hydroxylamine, indicating that collagenase contains an essential tyrosyl residue. In addition, acylation of the enzyme by diethylpyrocarbonate, diketene and acetic anhydride markedly lowers activity, which cannot be restored by hydroxylamine. This indicates that collagenase contains an essential lysyl residue, a conclusion supported by the fact that trinitrobenzene sulfonate also inactivates the enzyme.     

Identification of exposed surface glycoproteins in undifferentiated and differentiated mouse N-18 neuroblastoma cells

A simple method is described that permitted rapid isolation of plasma membranes from mouse N-18 neuroblastoma cells. The purified plasma membranes gave a 10-fold increase in the specific activity of incorporated [³H]fucose over that of the cell homogenate. The specific activities of two other membrane markers, 5′-nucleotidase and alkaline phosphatase, increased 11-fold and 15-fold, respectively. Metabolic labeling with [³H]fucose identified a major fucosyl glycoprotein with apparent molecular weight of 92 000. Three surface labeling methods together with SDS-polyacrylamide gel electrophoresis and fluorography were used to characterize and compare the surface glycoproteins of undifferentiated and differentiated N-18 cells. The galactose oxidase/NaB³H₄ method labeled two major galactoproteins (M_r = 52 000, 42 000) in both undifferentiated and differentiated cells. The neuraminidase/galactose oxidase/NaB³H₄ method revealed many sialylgalactoproteins. Among them, the 220-kdalton, 150-kdalton and 130-kdalton bands were at least 100% more prominently labeled in the differentiated calls whereas the 76-kdalton and 72-kdalton bands were less prominently labeled in the differentiated cells when compared to their undifferentiated counterparts. The prominently iodinated protein bands in the undifferentiated cells had apparent molecular weights of 130 000, 92 000, 76 000 and 72 000 as compared to 150-, 130-, 92- and 76-kdalton bands in the differentiated cells. The labeling data obtained will enable us to further study the changes of these identified surface glycoproteins, both quantitatively and topologically, during the differentiation of neuroblastoma cells.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with 125I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of 125I-labeled insulin to a polypeptide that gave an apparent Mr of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% beta-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 microM unlabeled insulin, but not by 1 microM unlabeled nerve growth factor. Using the same affinity labeling technique, 125I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an Mr 145 000 and an Mr 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with ¹²⁵I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of ¹²⁵I-labeled insulin to a polypeptide that gave an apparent M_r of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM unlabeled nerve growth factor. Using the same affinity labeling technique, ¹²⁵I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an M_r 145 000 and an M_r 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

DNA repair kinetics in irradiated undifferentiated and terminally differentiated cells

The brains of male Fisher 344 rats bearing 80-150 mg intracerebral 9L/Ro tumors were irradiated with doses of 1,250-5,000 rads of x- or gamma-rays. At various times after irradiation, the cerebellum and tumor were excised, dissociated into single cells and the DNA from these cells sedimented through alkaline sucrose gradients in zonal rotors with slow gradient reorienting capability. Quantitation of the DNA repair kinetics demonstrated that the process in both tumor cells and neurons has a fast and slow phase. Although all other alternatives cannot be completely eliminated, we suggest that these two phases are most reasonably interpreted as representing repair of lesions in very accessible and less accessible regions of the genome rather than 1) repair of different types of lesions such as single- or double-strand breaks or 2) removal of immediate breaks and breaks induced during excision repair of latent base damage. The slow repair phase is saturable, but not inducible in both tumor cells and neurons. The data suggest that tumor cells restore their chromosomal DNA structure to the unirradiated state faster than neurons because 1) they contain more of the repair system per unit of DNA and 2) a larger proportion of their genetic material is comprised of very accessible regions. The data also suggest that the entire tumor cell genome may be accessible to the repair enzyme(s), while it is possible that a portion of the neuronal genome may be completely inaccessible.     

Calcium currents of differentiated mouse neuroblastoma cells

Differentiated mouse neuroblastoma cells (line C 1300, clone N-18-TG₂A₁) were investigated by intracellular dialysis. A slow component was found in the potential-dependent inward current of these cells. The results of investigation of changes in amplitude of this component during variation of the ionic composition of the external and internal solutions showed that this component is due to transport of calcium ions. A calcium current was observed in all cells tested. The region of its activation was between –70 and –65 mV; maximal values of this current were reached when the membrane potential was between –30 and –40 mV. The kinetic characteristics of this current were examined. In its kinetics and potential-dependence, this calcium current of the mouse neuroblastoma cell membrane is analogous to the fast component of the calcium current in normal neurons of rat spinal ganglia.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 527–531, July–August, 1984.     

Interaction of concanavalin A with differentiated and undifferentiated murine neuroblastoma cells.

Tritium-labeled acetyl-concanavalin A (³H-Con A) was used to study its kinetics of binding at 0 °C to murine neuroblastoma cells (clone neuro 2-A) grown in the differentiated (monolayer) and Undifferentiated (spinner) states. The binding of ³H-Con A to both cell types gives sigmoidal saturation curves, suggesting positively cooperative binding of the lectin. The Hill coefficient is 1.75 for differentiated and 1.36 for Undifferentiated cells. The maximal number of ³H-Con A molecules bound per cell is 2.3 × 10⁷ and 3.4 × 10⁷ for differentiated and Undifferentiated cells, respectively, and the apparent rate constants for formation of the lectin-cell complex are 6.13 × 10², m^?1, s^?1 for the Undifferentiated and 6.68 × 10², m^?1, s^?1 for the differentiated cells. The lectin bound to spinner cells does not dissociate spontaneously to any measurable extent over a 60-min period at 0 or 37 °C, but the lectin-cell complex dissociates rapidly after addition of α-methyl-d-mannopyranoside. At 37 °C, this sugar causes virtually complete dissociation of the cell-lectin complex within 30 min. The ³H-Con A dissociated from spinner cells is indistinguishable from the original ³H-Con A by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, gel filtration through Bio-Gels P-30 and P-100, and specific binding to spinner cells. Both the original and the dissociated ³H-Con A are dimers at pH 7.4. The sugar-induced dissociation of the labeled lectin from spinner cells is not accompanied by shedding or inactivation of the lectin binding sites of the cell surface.     

Comparative analysis of some aspects of mitochondrial metabolism in differentiated and undifferentiated neuroblastoma cells

The aim of the present study is to clarify some aspects of the mechanisms of regulation of mitochondrial metabolism in neuroblastoma (NB) cells. Experiments were performed on murine Neuro-2a (N2a) cell line, and the same cells differentiated by all-trans-retinoic acid (dN2a) served as in vitro model of normal neurons. Oxygraphy and Metabolic Control Analysis (MCA) were applied to characterize the function of mitochondrial oxidative phosphorylation (OXPHOS) in NB cells. Flux control coefficients (FCCs) for components of the OXPHOS system were determined using titration studies with specific non-competitive inhibitors in the presence of exogenously added ADP. Respiration rates of undifferentiated Neuro-2a cells (uN2a) and the FCC of Complex-II in these cells were found to be considerably lower than those in dN2a cells. Our results show that NB is not an exclusively glycolytic tumor and could produce a considerable part of ATP via OXPHOS. Two important enzymes - hexokinase-2 and adenylate kinase-2 can play a role in the generation of ATP in NB cells. MCA has shown that in uN2a cells the key sites in the regulation of OXPHOS are complexes I, II and IV, whereas in dN2a cells complexes II and IV. Results obtained for the phosphate and adenine nucleotide carriers showed that in dN2a cells these carriers exerted lower control over the OXPHOS than in undifferentiated cells. The sum of FCCs for both types of NB cells was found to exceed significantly that for normal cells suggesting that in these cells the respiratory chain was somehow reorganized or assembled into large supercomplexes.     

A comparative analysis of the protein components of plasma membranes isolated from differentiated and undifferentiated mouse neuroblastoma cells in tissue culture.

The plasma membranes of the cells of mouse neuroblastoma clone NB41A, were isolated without fixation by hardening procedures and were characterised by their enzyme activities and by their morphology in the light and electron microscopes. The membranes were prepared from two kinds of differentiated monolayer cultures; one in which the cells were induced to form long axon-like processes by the addition of N6,O2'-dibutyryladenosine 3':5'-monophosphate to the culture medium, and the other in which the cells were induced to form processes by the addition of 5-bromo-2'-deoxyuridine. The proteins from the solubilised membranes were compared with similar preparation from the membranes of undifferentiated cells, grown in suspension, by sodium dodecylsulphate polyacrylamide gel electrophoresis, using the incorporation of radioactive amino acids and L-fucose into proteins in the cultures to follow the differences in the patterns of polypeptide synthesis and glycosylation of the membrane proteins. The differentiated cells induced with either inducer show an increased incorporation of L-fucose into two distinct components with molecular weights of 60 000 and 70 000. The two types of induced cells differ from each other in that N6,O2'-dibutyryladenosine 3':5'-monophosphate stimulates both glycosylation and protein synthesis, with a relative increase in the low molecular weight proteins, but 5-bromo-2'-deoxyuridine stimulates mostly increased glycosylation of the membrane proteins.     

Kinetics of enzyme-mediated reduction of lipid soluble nitroxide spin labels by living cells

Nitroxide spin labels can be reduced to the corresponding hydroxylamines in cells. The selective action of inhibitors, and thermal and chemical inactivation demonstrate that the reduction of nitroxides in cells is an enzymatic or enzyme-mediated process. The kinetics of reduction of doxylstearates are affected by the position of the doxyl moiety along the stearic acid chain. The doxyl moiety of 5-doxylstearate is close to the membrane surface, and its reduction is first order with respect to the nitroxide, whereas the doxyl moieties of 10- and 12-doxylstearate are in the membrane hydrocarbon region and their reduction is a zero-order process. The reduction of 16-doxylstearate which usually has a mixture of first- and zero-order kinetics becomes zero order with addition of an extracellular broadening agent, potassium trioxalatochromiate(III). These results suggest that the rate of reduction of doxyl moieties is controlled by their accessibility to reducing equivalents, i.e., the rate-limiting step for the reduction of the doxyl moiety deep in the membrane is the diffusion of reducing equivalents within or into the membrane. The reduction of doxylstearates in cells is inhibited by rotenone but not antimycin A, cyanide, propyl gallate or SKF-525A. It appears that the reduction of doxylstearates takes place at the level of the ubiquinone in the respiratory chain in mitochondria in these cells.     

Differences in the cyclic AMP-dependent phosphorylation of plasma membrane proteins of differentiated and undifferentiated L6 myogenic cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L6 myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L6 myoblasts was stimulated more than three fold by 5 x 10(-5) M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L6 cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L6 cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f2b. Therefore, the data show that differentiation in L6 cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

The growth and morphological characteristics of undifferentiated and differentiated cells of the F9 mouse teratocarcinoma line

Differentiation of the F9 cell line was induced by treating the cells with retinoic acid (10(-6) M) and dibutyryl cycloadenosinemonophosphate (10(-4) M). The population doubling time and the portion of cells in G1-phase increase and saturation density falls as the result of this treatment. Differentiated F9 cells demonstrate a decreased capacity of forming colonies in the soft agar, lose their capacity of proliferating at the clonal density, and acquire the limited life-span in culture after reseeding at a high density. Some cells in the differentiated population retain their capacity of forming colonies in the soft agar and (or) of binding antibodies against the stem cell marker SSEA-1. Cells with the stem cell morphology were found in the course of passaging of differentiated cells after reseeding at a high density. These cells were able to differentiate after the standard procedure of the induction of differentiation with retinoic acid and dibutyryl cAMP. Causes of the rising and supporting of heterogeneity of the differentiated F9 cells are discussed.     

Changes induced by apamin from bee venom on differentiated mouse neuroblastoma cells in culture

The minor active component of bee venom was applied to mouse neuroblastoma cultures. The cytological changes observed are reported. After 8-10 h of incubation with 5 micrograms/ml of apamin in the culture medium, considerable retractions of the processes are apparent. Electron microscopically, the alterations seen are predominantly found in the subcellular organelles. The peculiar configuration of the endoplasmic reticulum (ER) is striking. Concentric whorls of cisternae seem to engulf the remaining ground substance. Following a 24-hour incubation with 5 micrograms/ml of apamin, the cell membrane disintegrates. A deeply infolded nucleus, vacuoles and remnants of cell organelles are present. The previously intact synapses are totally degenerated. Similar experiments using lower concentrations of apamin do not depict any apparent changes either light or electron microscopically.