phosphorus-radioisotopes and Neuroblastoma

In 1981, I made the strong statement that "The therapeutic approach of internally administered radiopharmaceuticals offers the potential to outmode the present approaches of conventional radiation therapy and chemotherapy." The present article updates and further supports this statement with new data, especially with the use of [131I]MIBG in several cancers and proposes a plan for application of nonsealed source radionuclide therapy to most solid tumors.

Topics: 3-Iodobenzylguanidine; Adrenal Gland Neoplasms; Adult; Antibodies, Neoplasm; Brachytherapy; Child; Child, Preschool; Humans; Iodine Radioisotopes; Iodobenzenes; Middle Aged; Neoplasms; Neuroblastoma; Pheochromocytoma; Phosphorus Radioisotopes; Radioisotopes; Radiotherapy Dosage; Risk

Achieving local control is a crucial component in the management of neuroblastoma, but this may be complicated in the setting of prior radiation treatment, especially when the therapeutic target is in proximity to critical structures such as the spinal cord. The authors describe a pediatric patient with multiply recurrent neuroblastoma and prior high-dose radiation therapy to the spine who presented with progressive epidural disease. The patient was managed with resection and intraoperative high-dose-rate brachytherapy using a phosphorus-32 ((32)P) plaque previously developed for the treatment of brain and spine lesions.

Topics: Brachytherapy; Child; Epidural Space; Female; Humans; Magnetic Resonance Imaging; Neoplasm Recurrence, Local; Neuroblastoma; Neurosurgical Procedures; Phosphorus Radioisotopes; Radiotherapy Dosage; Spinal Cord Compression; Thoracic Vertebrae; Treatment Outcome

Ellipticine and doxorubicin are antineoplastic agents, whose action is based mainly on DNA damage such as intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts. The key target to resolve which of these mechanisms are responsible for ellipticine and doxorubicin anticancer effects is the development of suitable methods for identifying their individual DNA-damaging effects. Here, the (32)P-postlabeling method was tested to detect covalent DNA adducts formed by ellipticine and doxorubicin.. The standard procedure of (32)P-postlabeling assay, this procedure under ATP-deficient conditions, the version using extraction of adducts with n-butanol and the nuclease P1 enrichment version were used to analyze ellipticineand/ or doxorubicin-derived DNA adducts.. Two covalent ellipticine-derived DNA adducts, which are associated with cytotoxicity of ellipticine to human UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines, were detected by the (32)P-postlabeling method. These adducts are identical to those formed by the ellipticine metabolites, 13-hydroxy- and 12-hydroxyellipticine. In contrast, no covalent adducts formed by doxorubicin in DNA of these neuroblastoma cells and in DNA incubated with this drug and formaldehyde in vitro were detectable by the (32)P-postlabeling assay.. The results presented in this paper are the first to demonstrate that in contrast to covalent DNA adducts formed by ellipticine, the adducts generated by formaldehyde-mediated covalent binding of doxorubicin to DNA are not detectable by the (32)P-postlabeling assay. No DNA adducts were, detectable either in vitro, in incubations of DNA with doxorubicin or in DNA of neuroblastoma cells treated with this drug. The results also suggest that covalent binding of ellipticine to DNA of UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines is the predominant mechanism responsible for the cytotoxicity of this drug. To understand the mechanisms of doxorubicin anticancer effects on neuroblastoma cells, development of novel methods for identifying covalent doxorubicin-derived DNA adducts is the major challenge for further research.

Topics: Antineoplastic Agents; Cell Line, Tumor; DNA Adducts; Doxorubicin; Ellipticines; Humans; Isotope Labeling; Neuroblastoma; Phosphorus Radioisotopes

A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 G-protein activator (NG-GPA)) increased [(35)S]guanosine 5'-O-(thiotriphosphate) ([(35)S]GTPgammaS) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Galpha(o). The NG-GPA also increased GTPgammaS binding to purified, recombinant Galpha(i2), Galpha(i3), and Galpha(o), but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgammaS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT(1)-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT(1)-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgammaS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G(i)/G(o) proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G(i)/G(o) signaling systems.

Topics: Animals; Brain; Carrier Proteins; Cattle; Cell Line; Cell Membrane; Colforsin; Glioma; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Isoproterenol; NAD; Neuroblastoma; Pertussis Toxin; Phosphorus Radioisotopes; Tumor Cells, Cultured

The mechanism whereby agonist occupancy of muscarinic cholinergic receptors elicits an increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin has been examined. Addition of oxotremorine-M to SH-SY5Y neuroblastoma cells resulted in rapid increases in the phosphorylation of FAK (t(1/2) = 2 min) and paxillin that were independent of integrin-extracellular matrix interactions, cell attachment, and the production of phosphoinositide-derived second messengers. In contrast, the increased tyrosine phosphorylations of FAK and paxillin were inhibited by inclusion of either cytochalasin D or mevastatin, agents that disrupt the cytoskeleton. Furthermore, phosphorylation of FAK and paxillin could be prevented by addition of either wortmannin or LY-294002, under conditions in which the synthesis of phosphatidylinositol 4-phosphate was markedly attenuated. These results indicate that muscarinic receptor-mediated increases in the tyrosine phosphorylation of FAK and paxillin in SH-SY5Y neuroblastoma cells depend on both the maintenance of an actin cytoskeleton and the ability of these cells to synthesize phosphoinositides.

Topics: Actins; Androstadienes; Cell Adhesion; Cell Adhesion Molecules; Chromones; Cytochalasin D; Cytoskeletal Proteins; Cytoskeleton; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; GTP-Binding Proteins; Humans; Lovastatin; Morpholines; Muscarinic Agonists; Neuroblastoma; Nucleic Acid Synthesis Inhibitors; Paxillin; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Protein-Tyrosine Kinases; Receptor, Insulin; Receptors, Muscarinic; Second Messenger Systems; Tumor Cells, Cultured; Tyrosine; Wortmannin

We have analysed poly(ADP-ribose) glycohydrolase, the enzyme responsible for in vivo degradation of ADP-ribose polymers, by means of a biochemical assay based on the capacity of the enzyme to use a synthetic 32P-labelled polymer as a substrate. The visualization of the reaction has been achieved by separation of poly and mono(ADP-ribose) by thin-layer chromatography followed by autoradiography, whereas polymer hydrolysis has been quantified by counting the spots corresponding to poly and mono(ADP-ribose). By addition of the enzyme inhibitor ethacridine to the reaction mixture, we have confirmed the specificity of the procedure we have developed. The protocol has been applied to study the specific activity of glycohydrolase in nuclear extracts from different mammalian cell lines and to an apoptotic experimental system, namely HL60 cells treated with etoposide. We have observed the activation of the enzyme after a two-hour drug treatment, that is concomitant with the activation of poly(ADP-ribose) polymerase, the enzyme which synthesizes the polymer. These data suggest a precise regulation of ADP-ribosylation process during cell death by apoptosis.

Topics: Adenosine Diphosphate Ribose; Animals; Autoradiography; Cell Nucleus; CHO Cells; Chromatography, Thin Layer; Cricetinae; Ethacridine; Etoposide; Glycoside Hydrolases; HeLa Cells; HL-60 Cells; Humans; Kinetics; NAD; Neuroblastoma; Phosphorus Radioisotopes; Tumor Cells, Cultured

The localization and phosphorylation state of tau in LA-N-5 neuroblastoma cells was examined. Our results demonstrate that there are two populations of tau in LA-N-5 cells: cytosolic tau and nuclear tau. Indirect immunofluorescent microscopy revealed that nuclear tau is specifically localized to the nucleolus while cytosolic tau is diffusely distributed. To localize and quantitate tau in LA-N-5 cells by subcellular fractionation, a method was developed to extract tau from the nucleus while preserving the endogenous state of the protein. These studies revealed that 16% of the total tau protein in LA-N-5 cells is located in the nucleus and more specifically was found predominantly in the chromatin fraction containing DNA, chromatin, and associated proteins. The phosphorylation state of nuclear and cytosolic tau was examined by labeling LA-N-5 cells with 32Pi and immunoprecipitating tau from the different fractions. These data demonstrated that nuclear tau and cytosolic tau are phosphorylated approximately to the same extent. To determine if the phosphorylation of nuclear tau occurs in the nucleus, LA-N-5 nuclei were isolated, incubated with [gamma-32P]ATP, extracted, and tau was immunoprecipitated. Although numerous nuclear proteins were 32P-labeled, tau was not phosphorylated. These results suggest that nuclear tau is not phosphorylated in the nucleus but rather in the cytosol prior to transport into the nucleus. The specific localization of nuclear tau strongly suggests that it has a functional role in the nucleus. However, further studies are necessary to determine the function of nuclear tau and how it may be regulated by phosphorylation.

Topics: Autoradiography; Cell Line; Cell Nucleolus; Cell Nucleus; Cytosol; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; Humans; Immunoblotting; Microscopy, Fluorescence; Neuroblastoma; Phosphates; Phosphorus Radioisotopes; Phosphorylation; tau Proteins; Tumor Cells, Cultured

2-5A Synthetase and 2-5A phosphodiesterase were determined by analytical capillary isotachophoresis in comparison to radioenzymatic methods. By means of isotachophoretic analysis, a frequently used radioenzymatic 2-5A synthetase assay was optimized and the results of both assays were compared. Using the isotachophoretic assay the influence of interferon-related cytokines (tumor necrosis factor-alpha and interleukin-2) on 2-5A synthetase induction in neuroblastoma cells was estimated. In contrast to mononuclear blood cells, the tumor necrosis factor induced 2-5A synthetase in these cells. 2-5A Phosphodiesterase was determined using an isotachophoretic assay and a radioenzymatic method. Degradation of A2'p5'A2'p5'A (trimeric form of 2-5A core) was measured by isotachophoresis whereas degradation of a mixture of phosphorus-32 labeled 2-5A cores was registered by radioenzymatic assay. Activity of 2-5A phosphodiesterase was only insignificantly enhanced by interferon in mononuclear blood and neuroblastoma cells. In contrast to the radioenzymatic assays, an accurate determination of 2-5A synthetase as well as of 2-5A phosphodiesterase is possible using the isotachophoretic method because the reactions are followed by measuring the substrates ATP and A2'p5'A2'p5'A, respectively.

Topics: 2',5'-Oligoadenylate Synthetase; Alkaline Phosphatase; Capillary Action; Cell Line; Cytokines; Electrophoresis; Exoribonucleases; Humans; Interleukin-2; Leukocytes, Mononuclear; Neuroblastoma; Phosphorus Radioisotopes; Tumor Necrosis Factor-alpha

We investigated tyrosine kinase activity of the ret proto-oncogene products (proto-Ret proteins), using a cell lysate of NB-39-nu neuroblastoma cells. The 150 kDa and 170 kDa proto-Ret proteins immunoprecipitated with antibodies against their carboxy-terminal 20 amino acids were shown to be phosphorylated predominantly on tyrosine residues in immunocomplex kinase assay. The level of tyrosine phosphorylation of the 150 kDa proto-Ret protein was approximately 10-fold higher than that of the 170 kDa proto-Ret protein, although both proteins were expressed at similar levels in neuroblastoma cells. This result was confirmed by using a lysate of SK-N-MC human primitive neuroectodermal tumor cells transfected with the ret proto-oncogene. The kinase activity of proto-Ret proteins was significantly inhibited by antibodies against their kinase domain, indicating that these antibodies recognize crucial epitopes for the enzymatic activity. On the other hand, the proto-Ret proteins were not phosphorylated in vivo in NB-39-nu cells and SK-N-MC transfectants.

Topics: Amino Acids; Blotting, Western; Drosophila Proteins; Humans; Molecular Weight; Neuroblastoma; Phosphates; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Proto-Oncogenes; Receptor Protein-Tyrosine Kinases; Tumor Cells, Cultured; Tyrosine

Pleiotrophin (PTN) is a heparin-binding cytokine that functions as a neurite outgrowth promoting and mitogenic activity in vitro. PTN is highly conserved and its gene is widely expressed in mammalian tissues during development, suggesting important roles of PTN in vivo. However, the mechanisms by which PTN mediates its functional activities are unknown. We now report that an approximately 200 kDa (p200) protein is phosphorylated (pp200) in PTN stimulated NIH 3T3 and NB41A3 cells five minutes after stimulation with PTN. Phosphorylation is maximum at 15 minutes and is PTN concentration dependent. pp200 is recognized by antiphosphotyrosine antibodies and contains both phosphotyrosine and phosphoserine by phosphoamino acid analysis. The results suggest that PTN functions in part through activation of protein kinase(s) in NIH 3T3 and NB41A3 cells.

Topics: 3T3 Cells; Animals; Antibodies, Monoclonal; Autoradiography; Carrier Proteins; Cytokines; Electrophoresis, Polyacrylamide Gel; Mice; Mitogens; Molecular Weight; Neuroblastoma; Phosphates; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Phosphotyrosine; Tumor Cells, Cultured; Tyrosine

A 116 kDa protein in NG108-15 homogenates is labelled in the presence of [32P]NAD+. This protein was found to be poly(ADP-ribosyl)ated and appears to be a poly(ADP-ribosyl)transferase which has poly(ADP-ribosyl)ated itself. Sodium nitroprusside, an NO generating agent, also stimulates the labelling of this protein by [32P]NAD+, but this can only be seen in the presence of thymidine, which inhibits poly(ADP-ribosyl)transferase activity. Sodium nitroprusside also stimulates the labelling of this protein by [3H-nicotinamide]NAD+, indicating that NO facilitates the formation of an adduct between this protein and NAD+. The insensitivity of the linkage between the protein and NAD+ to mercuric ions indicates that the adduct does not involve thiol groups.

Topics: Animals; Electrophoresis, Polyacrylamide Gel; Glioma; Hybrid Cells; Kinetics; Mice; Molecular Weight; NAD; Neuroblastoma; Nitroprusside; Phosphorus Radioisotopes; Poly(ADP-ribose) Polymerases; Rats; Tumor Cells, Cultured

Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These alpha subunit proteins were Gi2 alpha, two isoforms of Gi3 alpha, and two isoforms of Go alpha. The G proteins that interacted with delta-opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G alpha subunits, these three pertussis toxin substrates were identified as Gi2 alpha, Go2 alpha, and one isoform of Gi3 alpha, which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the delta-opioid receptor in these hybrid cells by the observation that delta agonists D-Ala2-D-Leu5-enkephalin (DA-DLE) and D-Pen2-D-Pen5-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the mu agonist PL017 and the kappa agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC50 (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the Kd value of 10 +/- 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [32P]ADP-ribose into all three substrates decreased 35-83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Analgesics; Animals; Autoradiography; Cell Line; Cell Membrane; Cholera Toxin; Electrophoresis, Polyacrylamide Gel; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Glioma; GTP-Binding Proteins; Hybrid Cells; Kinetics; Mice; Molecular Weight; Neuroblastoma; Pertussis Toxin; Phosphorus Radioisotopes; Rats; Receptors, Opioid; Receptors, Opioid, delta; Virulence Factors, Bordetella

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.

Topics: Acetophenones; Amino Acid Sequence; Arachidonic Acids; Calcium; Cell Membrane; Cell Membrane Permeability; Digitonin; Diglycerides; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Molecular Sequence Data; Myelin Basic Protein; Neuroblastoma; Phospholipases A; Phospholipases A2; Phosphorus; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Quinacrine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Pertussis toxin (PTX) catalyzes the ADP-ribosylation of the alpha-subunit of GTP-binding proteins (G-proteins) in the presence of NAD+. Pertussis toxin also decreases the electrophoretic mobility of the alpha-subunit on urea SDS PAGE. This effect of PTX has been suggested to be a property of the toxin different from its ability to catalyze ADP-ribosylation. However, the present report provides evidence to the contrary; ie, this mobility shift required the ADP-ribosylation of alpha-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD+ (0.026-1.3 microM), there was a linear increase in the formation of the slower migrating alpha-subunit as measured by immunoblotting with selective antisera, (2) addition of NADase to the incubation mixture completely eliminated the formation of this protein, and (3) increasing concentrations of nicotinamide (50-250 mM), which inhibits ADP-ribosylation, decreased the amount of the slower migrating alpha-subunit. Thus, in addition to PTX, NAD+ was required for the mobility shift and the slower migrating alpha-subunit is likely the ADP-ribosylated form.

Topics: Adenosine Diphosphate Ribose; Animals; Autoradiography; Cell Line; Electrophoresis, Polyacrylamide Gel; Glioma; GTP-Binding Proteins; Hybrid Cells; Immunoblotting; Kinetics; Macromolecular Substances; NAD; Neuroblastoma; Niacinamide; Pertussis Toxin; Phosphorus Radioisotopes; Virulence Factors, Bordetella

The treatment of primary proliferative polycythaemia (polycythaemia rubra vera) may include radioactive phosphorus (P32) in conjunction with venesection. Acute leukaemia or carcinoma can be associated with the use of P32. We present a case of primary proliferative polycythaemia treated by repeat venesection together with P32 whose follow-up was complicated by the development of malignant neuroblastoma.

Topics: Bloodletting; Humans; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Neuroblastoma; Phosphorus Radioisotopes; Polycythemia Vera

Topics: Animals; Autoradiography; Cell Line; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Neuroblastoma; Phosphatidylinositols; Phosphorus Radioisotopes; Radioligand Assay; Tritium; Type C Phospholipases

1. Prostacyclin and adenosine A2 receptors activate adenylate cyclase in the neuroblastoma hybrid cell lines NG108-15 and NCB-20. Prolonged exposure of NG108-15 cells to iloprost (a stable analogue of prostacyclin) results in a subsequent reduction in the capacity for adenylate cyclase activation by iloprost, the adenosine analogue 5'-(N-ethyl)-carboxamidoadenosine (NECA) or NaF. In contrast prolonged exposure of NCB-20 cells to iloprost results only in the loss of iloprost responsiveness. 2. Iloprost pretreatment of NG108-15 cells also magnified the morphine-dependent inhibition of iloprost-stimulated adenylate cyclase activity from 36 to 48%. This change was not due to lower iloprost stimulation following desensitization, since the % inhibition of adenylate cyclase activity by morphine in control cells was constant irrespective of enzyme activity. 3. These heterologous effects observed in NG108-15 cells following iloprost pretreatment may involve changes in the GS alpha protein, since there was a reduction of about 30% in the cholera toxin-induced [32P]-ADP-ribosylation of a 45 kDa protein from cell membranes (corresponding to the extent of loss of NECA or NaF responsiveness). A similar reduction was not observed in NCB-20 cells. 4. These results indicate that iloprost pretreatment induces different forms of desensitization in NG108-15 and NCB-20 cell lines. The heterologous desensitization in the former may, like the human platelet, involve a functional loss of GS alpha from the cell membrane. Changes in the activity of GS alpha may also account for the heterologous effects on receptors that mediate inhibition of adenylate cyclase.

Topics: Adenosine; Adenosine Diphosphate; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cell Line; Epoprostenol; GTP-Binding Proteins; Hybrid Cells; Iloprost; Kinetics; Mice; Morphine; Neuroblastoma; Phosphorus Radioisotopes; Receptors, Epoprostenol; Receptors, Prostaglandin; Sodium Fluoride

Adult rats were intracraneally injected with [32P] phosphate and brain microtubules isolated. The electrophoretically purified, in vivo phospholabeled, beta-tubulin was digested with the V8-protease and the labeled peptide purified by reversed-phase liquid chromatography. Its amino acid sequence corresponds to the COOH-terminal sequence of a minor neuronal beta 3-tubulin isoform from chicken and human. The phosphorylation site was at serine 444. A synthetic peptide with sequence EMYEDDEEESESQGPK, corresponding to that of the COOH terminus of beta 3-tubulin, was efficiently phosphorylated in vitro by casein kinase II at the same serine 444. The functional meaning of tubulin phosphorylation is still unclear. However, the modification of the protein takes place after microtubule assembly, and phosphorylated tubulin is mainly present in the assembled microtubule protein fraction.

Topics: Amino Acid Sequence; Animals; Brain; Casein Kinases; Cell Differentiation; Cell Line; Chromatography, High Pressure Liquid; Humans; Kinetics; Microtubules; Molecular Sequence Data; Neuroblastoma; Neurons; Peptide Fragments; Peptide Mapping; Peptides; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Radioisotope Dilution Technique; Rats; Sequence Homology, Nucleic Acid; Tubulin

The phosphorylation of rat brain microtubule protein on intracranial injection of labeled phosphate has been analyzed. The major microtubule protein components phosphorylated in vivo in rat brain are the high-molecular-weight microtubule-associated proteins (MAPs) MAP-1A, MAP-1B, and MAP-2. A slight phospholabeling of beta-tubulin, which corresponds to the phosphorylation of a minor neuronal beta-tubulin isotype, is also observed. Whereas MAP-1B, MAP-2, and beta-tubulin are phosphorylated in the brain of 5-day-old rat pups, when most neurons of the CNS are extending processes, MAP-1A phosphorylation is observed only after neuronal maturation takes place. The phosphorylation of MAP-1A, MAP-1B, and beta-tubulin may be due mainly to casein kinase II or a related enzyme, whereas MAP-2 appears to be modified by other enzymes such as the cyclic AMP-dependent protein kinase (protein kinase A) and the calcium/phospholipid-dependent protein kinase (protein kinase C). Microtubule protein phosphorylation has also been studied in neuronal cultures. In differentiated neuroblastoma cells, only MAP-1B and beta-tubulin are phosphorylated in a manner coupled to neurite outgrowth. In primary cultures of fetal rat brain neurons, the pattern of microtubule protein phosphorylation resembles that found in vivo in rat pup brain. As phosphorylated MAP-1A and MAP-1B are present mainly on assembled microtubules, whereas the phosphorylation of MAP-2 decreases its interaction with microtubules, a role can be suggested for the phosphorylation of these proteins in the regulation of microtubule assembly and disassembly during neuronal development.

Topics: Aging; Animals; Brain; Cell Line; Cells, Cultured; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Fetus; Macromolecular Substances; Male; Mice; Microtubule Proteins; Microtubule-Associated Proteins; Neuroblastoma; Neurons; Peptide Mapping; Phosphates; Phosphopeptides; Phosphorus Radioisotopes; Phosphorylation; Rats; Rats, Inbred Strains; Trypsin; Tubulin

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cell Line; Kinetics; Membrane Lipids; Mice; Neuroblastoma; Phosphates; Phospholipids; Phosphorus Radioisotopes; Phosphorylation; Sodium; Tritium

A method based on in situ hybridization, autoradiography and transmission electron microscopy for mapping genes on human metaphase chromosomes is presented. Successful mapping of the tandemly repeated rDNA genes and of two nucleic acid probes, N-myc and probe 3 (Kanda et al. 1983), that are amplified in a homogeneously staining region (HSR) of the neuroblastoma cell line, IMR-32 is described. By using sufficiently thin AgBr emulsions, it is possible to obtain observable grains and good resolution with probes radiolabeled with 3H, 35S, or 32P, but the former gives the best results. We observe that neither of the two probes, N-myc and probe 3, has a uniform spatial distribution along the HSR and that the distributions of the two probes differ from each other. These observations support previous studies which indicated that the formation of an HSR is a more complex process than uniform amplification of a single DNA segment to form an n-fold set of perfect tandem repeats. The present study shows that the electron microscopic method is useful for extending the results of light microscopic studies for problems where higher resolution mapping is needed.

Topics: Cell Line; Cells, Cultured; Chromosome Mapping; Chromosomes, Human; Genes; Humans; Lymphocytes; Metaphase; Microscopy, Electron; Mitosis; Neuroblastoma; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Sulfur Radioisotopes; Tritium

The phosphorylation of H1 histone subfractions was measured in mouse neuroblastoma cells stopped from dividing by three treatments that block cell division: 5 mM butyrate, 2% dimethyl sulfoxide, and serum withdrawal. H1 histone phosphorylation decreased in response to all three treatments, but the response differed in its timing and its extent for the different H 1 subfractions. The different decreases in phosphorylation correlated well with the differential decreases in biosynthesis of the individual H1 subfractions; however, an exception to this parallel decrease in synthesis and phosphorylation was observed in the case of histone H1(0). Phosphorylation of H1(0) was absent in each of the three treatments after 2 days, despite the continued synthesis and deposit of H1(0) on the chromatin. Thus, despite the fact that H1(0) was being synthesized and that the other newly synthesized H1 subfractions were phosphorylated at this time, the phosphorylation of H1(0) became uncoupled from its synthesis after prolonged treatments blocking cell division.

Topics: Animals; Cell Division; Cell Line; Chromatin; Electrophoresis, Polyacrylamide Gel; Histones; Kinetics; Mice; Molecular Weight; Neuroblastoma; Phosphates; Phosphorus Radioisotopes; Phosphorylation

Hydroxylamine stability has been used to classify (ADP-ribose)protein bonds into sensitive and resistant linkages, with the former representing (ADP-ribose)glutamate, and the latter, (ADP-ribose)arginine. Recently, it was shown that cysteine also serves as an ADP-ribose acceptor. The hydroxylamine stability of [cysteine([32P]ADP-ribose)]protein and [arginine([32P] ADP-ribose)]protein bonds was compared. In transducin, pertussis toxin catalyzes the ADP-ribosylation of a cysteine residue, whereas choleragen (cholera toxin) modifies an arginine moiety. The (ADP-ribose)cysteine bond formed by pertussis toxin was more stable to hydroxylamine than was the (ADP-ribose)arginine bond formed by choleragen. The (ADP-ribose)cysteine bond apparently represents a third class of ADP-ribose bonds. Pertussis toxin ADP-ribosylates the inhibitory guanyl nucleotide-binding regulatory protein (Gi) of adenylate cyclase, whereas choleragen modifies the stimulatory guanyl nucleotide-binding regulatory protein (Gs). These (ADP-ribose)protein linkages are identical in stability to those formed in transducin by the two toxins, consistent with the probability that cysteine and arginine are modified in Gi and Gs, respectively. Bonds exhibiting differences in hydroxylamine-stability were found in membranes from various non-intoxicated mammalian cells following incubation with [32P]NAD, which may reflect the presence of endogenous NAD:protein-ADP-ribosyl-transferases.

Topics: Adenosine Diphosphate Ribose; Animals; Cattle; Cell Line; Cell Membrane; Glioma; GTP-Binding Proteins; Hybrid Cells; Hydroxylamine; Hydroxylamines; Kinetics; Mice; Neuroblastoma; Nucleoside Diphosphate Sugars; Phosphorus Radioisotopes; Rats; Retina; Structure-Activity Relationship

Topics: Adrenal Gland Neoplasms; Biopsy; Ethanolamines; Female; Humans; Infant, Newborn; Liver; Liver Neoplasms; Magnetic Resonance Spectroscopy; Neuroblastoma; Phosphocreatine; Phosphorus Radioisotopes

The compound ppp(A2'p)3A3'[32P]pCp is a commercially available radioactive analogue of the 2',5' oligoadenylate series ppp(A2'p)nA, n greater than or equal to 2, commonly referred to as 2-5A. It is used as a probe for measuring concentrations in competition radiobinding and radioimmune assays. We have found that incubation of the probe with extracts from HeLa, CV1, or neuroblastoma cells results in its covalent attachment to two size classes of RNA: the first includes a major species with a molecular weight of approximately 350,000, the second is much smaller (40 +/- 5 nucleotides in length) and could represent tRNA half-molecules. Ligation is to the 3' end of the probe molecule with formation of a 3',5'-phosphodiester bond. Thus, probe ligation provides a sensitive and convenient assay for the detection not only of RNA ligase(s) but also of ligatable RNAs (such as the putative tRNA half-molecules) in mammalian cell extracts.

Topics: Animals; Cell Line; Chlorocebus aethiops; Electrophoresis, Polyacrylamide Gel; HeLa Cells; Humans; Kidney; Kinetics; Neuroblastoma; Oligonucleotides; Oligoribonucleotides; Phosphorus Radioisotopes; Polynucleotide Ligases; RNA Ligase (ATP); RNA, Neoplasm

Topics: Cell Line; Chromatography, Thin Layer; Neuroblastoma; Phospholipids; Phosphorus Radioisotopes

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.

Topics: Adenine; Adenine Nucleotides; Adenosine; Adenosine Monophosphate; Adenylyl Cyclases; Animals; Clone Cells; Cytidine; Guanine; Guanosine; Kinetics; Mice; Neuroblastoma; Phosphorus Radioisotopes; Prostaglandins E; Ribose; Theophylline; Tolazoline; Uridine; Yohimbine

Topics: Adenosine Triphosphatases; Adenylyl Cyclases; Amidohydrolases; Animals; Carcinoma; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Clone Cells; Female; Fibroblasts; Guinea Pigs; HeLa Cells; Humans; Kinetics; Laryngeal Neoplasms; Liver; Mice; Naphthols; Nasopharyngeal Neoplasms; Neuroblastoma; Nucleotidases; Phosphoric Diester Hydrolases; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Pyrophosphatases; Uterine Cervical Neoplasms

Topics: Carbon Radioisotopes; Cell Fractionation; Cell Line; Cells, Cultured; Clone Cells; Cytoplasm; Kinetics; Lithium; Methionine; Neuroblastoma; Phosphates; Phosphorus Radioisotopes; Ribosomes; RNA, Ribosomal; Spectrophotometry; Spectrum Analysis; Tritium; Uracil Nucleotides; Uridine