Neuroblastoma Lines Research Articles

Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD.

MyoD is a master regulatory gene for myogenesis. Under the control of a retroviral long terminal repeat, MyoD was expressed in a variety of differentiated cell types by using either a DNA transfection vector or a retrovirus. Expression of muscle-specific proteins was observed in chicken, human, and rat primary fibroblasts and in differentiated melanoma, neuroblastoma, liver, and adipocyte lines. The ability of MyoD to activate muscle genes in a variety of differentiated cell lines suggests that no additional tissue-specific factors other than MyoD are needed to activate the downstream program for terminal muscle differentiation or that, if such factors exist, they are themselves activated by MyoD expression.

Purification and characterization of glia maturation factor beta: a growth regulator for neurons and glia.

A protein has been isolated from bovine brains by using a modification of the procedure used to purify glia maturation factor. The method consists of ammonium sulfate precipitation, chromatography with DEAE-Sephacel, Sephadex G-75, and hydroxylapatite columns, passage through a heparin-Sepharose column, and finally fractionation by reverse-phase HPLC with a C4 column. The isolated protein reacts strongly with the mouse monoclonal antibody G2-09 and has a molecular weight of approximately 17,000 and an isoelectric point of pH 4.9. The N terminus is blocked, but tryptic digestion releases 28 peptides, 8 of which have been sequenced. The total known residues add up to more than two-thirds of the entire 140-residue protein, estimated from amino acid composition, and show no sequence homology with any known protein. Reversible thermal renaturation greatly enhances its biological activity. The purified protein stimulates differentiation of normal neurons as well as glial cells. It inhibits the proliferation of the N-18 neuroblastoma line and the C6 glioma line while promoting their phenotypic expression. We designate this protein glia maturation factor beta.

Neuroblastoma double minutes isolated by pulsed-field gel electrophoresis without prior strand-cleaving treatments

In a human neuroblastoma line, minute chromosomes were separable from the bulk of interphase nuclear DNA by contour-clamped homogeneous electric field (CHEF) gel elecyrophoresis. The minute chromosomes showed a homogeneous size of ∼3 Mbp and contained amplified N-myc genes. Fractionation was accomplished without prior strand-cleaving treatment of the DNa, indicating that at least a portion of the minute chromosomes exist as free entities in the interphase nuclei. Human alphoid satellite DNA sequences were also detected in the 3-Mbp band. It is possible that alphoid sequendes arecontained in the constricted central region that joins these double minutes.

Dideoxycytidine, an anti-HIV drug, selectively inhibits growth but not phosphatidylcholine metabolism in neuroblastoma and glioma cells

Dideoxycytidine (ddCyd), an inhibitor of AIDS-related HIV, has been examined for effects on cell proliferation and phosphatidylcholine synthesis in tumor lines of nervous system origin. Uptake and metabolism of [3H]ddCyd, observed in all cells, was greatest in one human neuroblastoma line, HTB-10. Growth of the HTB-10 line was markedly inhibited by 40 microM ddCyd, whereas growth of C6 glioma and N1E-115 or HTB-11 neuroblastoma cells was unaltered. Phosphatidylcholine synthesis in the presence or absence of stimulation by phorbol ester was not specifically altered by ddCyd. Thus, ddCyd was incorporated and inhibited growth in a cell-specific manner but had little effect on cytidine-dependent phospholipid synthesis. This suggests that some cells derived from the nervous system may be more susceptible than others with respect to the positive and negative effects of ddCyd as a potential antiviral drug.

The Effect of γ Radiation on DNA Methylation

The effect of 60Co gamma radiation on DNA methylation was studied in four cultured cell lines. In all cases a dose-dependent decrease in 5-methylcytosine was observed at 24, 48, and 72 h postexposure to 0.5-10 Gy. Nuclear DNA methyltransferase activity decreased while cytoplasmic activity increased in irradiated (10 Gy) V79A03 cells as compared to controls. No DNA demethylase activity was detected in the nuclei of control or irradiated V79A03 cells. Additionally, gamma radiation resulted in the differentiation of C-1300 N1E-115 cells, a mouse neuroblastoma line, in a dose- and time-dependent manner. These results are consistent with the hypothesis that (1) genes may be turned on following radiation via a mechanism involving hypomethylation of cytosine and (2) radiation-induced hypomethylation results from decreased intranuclear levels of DNA methyltransferase.

Lack of effect of the opioid antagonist, naltrexone, on the [formula omitted] growth of C-1300, NIE-115 and NS206 murine neuroblastoma tumor cell lines

Attempts have been made to confirm previously reported results which demonstrated that the opioid antagonist, naltrexone, altered the in situ growth of murine neuroblastoma tumors. Adult male A/J mice were injected with tumor cells from three different cell lines of murine neuroblastoma; the spontaneously arising C-1300 line, the adrenergic clonal line NIE-115, and the cholinergic clonal line NS20Y. Naltrexone was administered daily in doses of 0.1, 0.4, or 10.0 mg/kg subcutaneously, to replicate the reported experimental design. In contrast to previous studies, we were unable to demonstrate any effect of naltrexone on in situ growth or other characteristics of tumors produced by the C-1300, NIE-115 or NS20Y murine neuroblastoma cell lines. Ligand binding studies have demonstrated the presence of high levels of opiate binding sites on membranes prepared from the NS20Y clonal cell line and low levels on the membranes of the C1300 tumor line.

Nerve growth factor induces neurite outgrowth in a clone derived from an NGF-insensitive human neuroblastoma cell line

Nerve growth factor (NGF) plays a trophic and tropic role in the development of vertebrate sympathetic and sensory ganglia; however, the precise nature of the NGF effect(s) is not understood. Study of NGF-responsive human neuroblastoma cell lines allows characterization of NGF-induced neunte outgrowth in cells not dependent on NGF for survival. The human neuroblastoma line SK-N-SH did not significantly extend neuntes in response to NGF, but did show an increase in cell number. By contrast, a clone of the line, SH-IN, extended neuntes in response to NGF or dibutyryl cyclic AMP, and showed inhibition of cell proliferation. Thus, cells capable of morphological differentiation in response to NGF can be cloned from neuroblastoma cell lines in which the majority of the cells fail to extend neurites even in the presence of NGF.

Recognition of an in vivo immune response to human neuroblastoma modulation of antigen expression by retinoic acid.

Neuroblastoma is one of the most common solid tumors of childhood and is notable for its ability to spontaneously regress and, in some instances, to differentiate to less malignant ganglioneuromas. Since immune mechanisms may account for these phenomena, identification of in vivo immune responses to tumor cell surface antigens may be important to the progression of the disease. As determined by analysis on the fluorescence-activated cell sorter, sera from 10 of 18 neuroblastomas patients were found to contain antibodies to a cell surface antigen present on subpopulations of cells from human neuroblastoma cell lines maintained in vitro. Eight human neuroblastoma cell lines were examined and found to vary in reactivity with sera. Induction of differentiation of cell lines with retinoic acid (RA) in vitro resulted in most cell lines bearing higher percentages of positive cells but with a decreased mean cell fluorescence. Preliminary Western blot analysis of lysates of the human cell lines NMB/N7, SMS-KAN, and SK-N-MC showed two principal antigen bands on reducing gels. Comparison of sera from different individuals on lysates of cell lines showed reactivity principally with bands of 105-110 kD and 65-70 kD and an additional minor band of slightly lower molecular weight with the higher titer sera. The ability of different sera to recognize a common antigen pattern suggests that this represents an immunodominant cell surface antigen. Examination of reactivity of other cell lines in this system showed that positive sera reacted with all neuroblastoma lines examined, one neuroepithelioma (SK-N-MC), two melanoma lines (MeWo, G361), and one adrenal-derived adenocarcinoma (SW-13).

IFN-treated neuroblastoma cell lines remain resistant to T cell-mediated allo-killing, and susceptible to non-MHC-restricted cytotoxicity.

Neuroblastoma cell lines can have very low MHC Ag expression. The cell lines are insensitive to allo-killing by primed CTL, but are sensitive to non-MHC-restricted cytotoxicity. IFN-gamma increased class I expression, but the cells remained insensitive to CTL. Susceptibility to nonrestricted effectors was preserved. Class I+ glioma cell lines behaved similarly. The CTL resistance was localized to the recognition phase. Neuroblastoma lines did not form conjugates with primed T cells, but were lysed if they were coupled to the effectors via lectins. The levels of class I expression, and resistance to CTL, were constant over a range of IFN doses. HLA-A,B,C structure and distribution were studied more intensively on one cell line, CHP-100. HLA-A2 and -A3 were present on greater than or equal to 99% of the cells, in a unimodal distribution. After IFN treatment, the levels were similar to B cell controls. In two-dimensional gel electrophoresis, the molecules co-migrated with those of B cell controls. The defect may thus be in accessory proteins that are necessary for T cell recognition or binding, rather than in the structure or distribution of the HLA-A,B,C proteins.

Synapse-competence of LA-N-2 human neuroblastoma cells in coculture with rat striated muscle cells

The purpose of this study was to determine whether cells of the human neuroblastoma line. LA-N-2. are capable of establishing functional synapses in culture. We used a coculture system in which striated muscle cells from the rat served as postsynaptic targets for the cholinergic LA-N-2 cells. By recording postsynaptic responses from muscle cells, differentiated LA-N-2 cells were found to innervate muscle cells, releasing acetylcholine spontaneously at LA-N-2-muscle synapses. A subpopulation of the LA-N-2 cells forming synapses with the muscle cells also developed the ability to release acetylcholine in response to stimulation. This, coupled with results obtained from experiments examining the time course of synapse formation, led us to propose that the extent to which LA-N-2 cells in our coculture system are differentiated may vary and that this variation may underlie the degree to which they express neuron-like transmission properties.

Expression of markers shared between human natural killer cells and neuroblastoma lines.

Neuroblastoma is a tumor of neuroectodermal origin arising most commonly from the adrenal medulla. We have examined the ability of several monoclonal antibodies which recognize markers predominantly expressed on human natural killer (NK) cells to react with neuroblastoma cell lines in vivo derived sections of tumor. HNK-1 (Leu 7) is a monoclonal IgM antibody which recognizes a carbohydrate epitope on NK cells and a wide range of tumor cell types. We have shown that HNK-1 recognizes the human neuroblastoma lines SMS-KCNR, SMS-KAN, NMB/N7, and IMR/5. Expression of this antigen on cell lines can be slightly increased by retinoic acid-induced differentiation of the cells. N901 (NKH1), a monoclonal antibody raised against interleukin 2-dependent human NK cell lines also recognizes all human neuroblastoma cell lines examined. This expression is independent of differentiation induction and levels remain unaltered following retinoic acid treatment of the cell lines. Lastly, with monoclonal antibody 49H.8, it has been found that reactivity of the lines is weak until induction of differentiation, after which highly significant increases of reactivity are seen. 49H.8 recognizes several cryptic carbohydrate antigens with varying affinities, shown to identify mouse and rat NK cells. In contrast to other NK markers, human neuroblastoma cell lines did not express significant reactivity with B73.1, Leu 11b, or Leu 18. Immunohistochemical staining of sections of human neuroblastoma tumors correlated with the in vitro findings; however, staining with N901 and 49H.8 was only seen on frozen sections, not paraffin-embedded. The significance of shared NK cell-neuroblastoma/neuron antigens is currently under investigation.

Role of adenosine 3′5′-cyclic monophosphate in antineoplastic effect of prostaglandins (PGE 1, PGE 2, PGD 2 and PGA 1) on human neuroblastoma cells

To determine the role of adenosine 3′5′-cyclic monophosphate (cAMP) in the antineoplastic effect of prostaglandins (PGE 1, PGE 2, PGD 2 and PGA 1), we studied 2 cell lines of human neuroblastoma; i.e. GOTO and SK-N-DZ. PGE 1 or E 2 at 30 μg/ml and PGD 2 or PGA 1 at 5 μg/ml were cytotoxic to these neuroblastoma cells. In both cell lines, increase of intracellular cAMP was closely associated with E-type PGs cytotoxicity, however, in PGD 2, or PGA 1 cytotoxicity, cAMP increase was observed only in GOTO cells. Pretreatment of GOTO cells with 5 μg/ml PGE 2 for 24 hr caused a desensitization of cAMP responses to PGE 1, PGD 2 or PGA 1 only in association with a reduced cytotoxicity of PGE 1. On the other hand, PGE 2-pretreated SK-N-DZ cells resulted in a desensitization in response to PGE 1, but not to other PGs, without affecting the cytotoxicity of these PGs. A decreased [ 3H]PGE 1 binding similarly occurred in either the PGE 2-pretreated GOTO or SK-N-DZ cells. However, cholera toxin- or forskolin-induced cAMP production was suppressed only in the pretreated GOTO cells. cAMP response by forskolin rather increased in the pretreated SK-N-DZ cells. These results indicate that antineoplastic effect of E type PGs mediates through cAMP, but not that of PGD 2 and PGA 1 and that PGE 2 pretreatment may cause a down regulation of PGE 1 receptor site in both cell lines. It is also suggested that PGE 2 pretreatment results in a heterologous desensitization in GOTO and a homologous desensitization in SK-N-DZ cells.

Chapter 9 Monoclonal antibodies directed against small cell carcinomas define by flow cytometry phenotypic differences among small cell and non-small cell carcinomas, neuroblastomas, and lymphoblastoid cell line

Abstract Indirect immunofluorescence and flow cytometry were used to determine reactivity of workshop antibodies with a large panel of human cultured cell lines which included small cell lung carcinoma (SCLC) “classic”, and “variant”; and non-small cell lung carcinomas (NSCLC). Other cell lines were SHP-77 1 (an unusual “oat cell” large cell variant which has the morphology of a “variant”, but biochemical properties of a “classic” SCLC). Also studied were 2 neuroblastoma lines, 2 colon carcinoma and 5 lymphoblastoid lines. Three morphologically similar “classic” SCLC were phenotypically different in reactivity with workshop antibodies, suggesting that testing with such a panel is a new means to classify SCLC. The “classic” lines also differed in intensity of labelling with some of the reactive monoclonals, indicating differences in density of surface markers. Xenografts of human tumour lines grown in nude mice reacted very strongly with (FAB 1 ) 2 anti-mouse fragments, suggesting that such xenografts have nude mouse immunoglobulins on their surface. Evidence is presented that some monoclonals may react with tumour cells via Fc receptors NCSCLC, colon carcinoma, and lymphoblastoid lines also react with some workshop antibodies.

Use of human neuroblastoma continuous cell lines for in vitro drug sensitivity screening.

We have used three continuous human neuroblastoma cell lines to establish patterns of in vitro drug sensitivities, as judged by clonogenic assay. We evaluated 12 'standard' antitumor drugs already in clinical usage, and tested four newer analogues, one of cisplatin and three of doxorubicin, and the investigational agent desferrioxamine. A certain heterogeneity of drug sensitivities was noted amongst these three cell lines, but a few general conclusions can be drawn. Responses of all lines tested were similar for actinomycin D, dibromodulcitol, doxorubicin, 5-fluorouracil, melphalan and VM 26. However, line CHP 100 proved hypersensitive to amsacrine, bleomycin, methotrexate and vincristine yet refractory to cisplatin, carboplatin and VP-16, compared with the other two lines. This emphasizes the necessity for using a panel of cell lines for this type of drug screening programme. A comparison of IC50 drug concentrations, derived from these in vitro tests, with plasma levels achievable clinically, indicate that VP-16, VM 26, doxorubicin and cisplatin appear to be the most effective agents in this tumor type. This finding is consistent with clinical experience. The newer doxorubicin analogues proved 2-5 fold more cytotoxic than doxorubicin itself. However, these differences also appear to be reflected in the lower dose ranges now being tested in phase I/II clinical trials. Desferrioxamine, which proved cytotoxic against all three neuroblastoma cell lines, exerted comparable cytotoxicity against two of the three non-neuroblastoma human tumor cell lines evaluated. Therefore we suggest that attempts to identify any specific antineuroblastoma activities by new investigational agents using this type of model systems require evaluation against panels of both neuroblastoma and non-neuroblastoma lines.

Comparison of epi-GM3 with GM3 and GM1 as stimulators of neurite outgrowth

A variety of naturally occurring ganglioside structures were previously shown to be effective agents for inducing neurite outgrowth of primary neurons and neuroblastoma lines. We report here the results of similar experiments with a synthetic epimer of GM3 (epi-GM3) possessing a neuraminidase-resistant β-ketosidic linkage. This substance was found to enhance neuritogenesis toward two transformed cell lines (neuro-2A, PC-12) and one primary neuronal tissue (dorsal root ganglia). The results indicate that the stereochemistry of the ketoside linkage is not critical and that metabolism of exogenous ganglioside by the treated cells is not involved directly in the neuritogenic phenomenon.

Pyrimidine base degradation in cultured murine C-1300 neuroblastoma cells and in situ tumors.

Dihydropyrimidine dehydrogenase (DPD), the initial, rate-limiting step in pyrimidine degradation, was studied in two cell lines of murine neuroblastoma (MNB-T1 and MNB-T2) that were derived from C-1300 MNB tumor carried in A/J mice. The MNB-T2 (low malignancy) cell line was originally derived from the in situ tumor and carried in tissue culture for more than 100 passages; the MNB-T1 (high malignancy) line consisted of a new sub-culture that was also established from the in situ MNB tumor. DPD activity was determined in cytosolic preparations of MNB utilizing high performance liquid chromatography to separate the radiolabeled substrate ([2-14C]thymine) from [2-14C]dihydrothymine. The apparent affinity of DPD for NADPH in MNB cells (Km approximately 0.08 mM) was identical to that of A/J mouse brain and liver. The DPD activity of the high malignancy (MNB-T1) cell line was 14.3% of that observed in the low malignancy (MNB-T2) line. In situ tumors formed after implantation of high malignancy (MNB-T1) cells into A/J mice had only 25.2% of the DPD activity observed in tumors derived from low malignancy (MNB-T2) cells. When MNB-T2 cells were injected into naive A/J mice, tumors developed in only 68% of animals, the tumor growth rate was slow and a mortality of 20% was observed. In contrast, tumors derived from injected MNB-T1 cells showed a faster growth rate and 100% mortality. Most MNB-T2 derived tumors were not lethal and ultimately resolved while the MNB-T1 derived tumors were invariably lethal. These studies support the concept that the levels of DPD activity in neoplastic cells are inversely related to their malignant expression and also provide a model to study differences between neuroblastoma cell lines derived from the same in situ tumor but which manifest different neoplastic behavior.

A cell line with decreased sensitivity to the methyl mercury-induced stimulation of α-amanitin sensitive RNA synthesis in isolated nuclei

1. 1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on α-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of α-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275–6279). 2. 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.

Common features of antigenic determinants present on mammalian nerve cell membrane structures and in cytoplasmic tetrodotoxin-sensitive proteins

Possible antigenic features common to nerve cell membrane structures and cytoplasmic tetrodotoxin-sensitive proteins were investigated by means of an immobilized immunoenzyme test. It was found that antiserum obtained by immunizing rabbits with a purified preparation of cytoplasmic tetrodotoxin-sensitive proteins can react with antigenic determinants present on the membrane fraction of bovine brain cells, on rat synaptosomes and on cells of a clonal line of mouse neuroblastoma. It was shown by means of an inhibition assay test that antibodies of the same specificity contribute to the observed response. Findings would indicate the presence of antigenic determinants common to nerve cell membrane structures and cytoplasmic tetrodotoxin-sensitive proteins. This is consistent with the hypothesis that cytoplasmic tetrodotoxin-sensitive proteins have certain features in common with membrane sodium channels.

Sorcin (V19), a soluble acidic calcium-binding protein overproduced in multidrug-resistant cells: Identification of the protein by anti-sorcin antibody

Sorcin (soluble resistance-related calcium-binding protein), an acidic (pI = 5.7) protein ( M r ∼ 20 kDa) previously designated V19, was originally identified in cells selected for high levels of resistance to vincristine. Two-dimensional gel electrophoresis and/or Western blot techniques now show sorcin to be overproduced in cells selected for resistance to actinomycin D (QUA/ADj), colchicine (CH RC5), and adriamycin (BE(2)-C/ADR). Not all cell lines selected for resistance to these drugs overproduced sorcin; e.g. cells of an independently selected actinomycin d-resistant subline of QUA, QUA/ADsx, did not contain increased amounts of sorcin. Sorcin was purified by preparative gel electrophoresis from QUA/ADj cells and used to generate specific antiserum in chickens. By Western blot analyses the antiserum was shown to recognize sorcin in QUA/ADj and in vincristine-resistant mouse and Chinese hamster lung, colchicine-resistant Chinese hamster ovary, and adriamycin-resistant human neuroblastoma lines. Low level expression of the protein was detectable in control, drug-sensitive cells. Direct binding assays with 45Ca 2+ showed that sorcin was a calcium-binding protein. QUA/ADj cells contained increased numbers of double minute chromosomes (DMs), cytogenetic indicators of gene amplification. As found for two other multidrug-resistant sublines, sorcin overproduction in QUA/ ADj cells may be the result of amplification of the sorcin-encoding gene. The overproduction of this protein in multidrug-resistant cells of various species implies that sorcin plays a role in expression of the resistant phenotype.

The expression and detection of MHC class I antigens on murine neuroblastoma and ependymoblastoma lines

It has been reported that human neuroblastoma lines are almost devoid of class I transplantation antigens, while human glioma lines express these antigens. Other studies have also shown a paucity of class I antigens on the murine neuroblastoma line N2A, and the expression of these antigens by the murine ependymoblastoma G26 lines. Such differences might represent heterogeneity in class I antigen expression by different brain cell types, and the importance of this to the immunology of the brain prompted us to re-examine class I expression by these cell lines in more detail. Using an exhaustive number of approaches, we were not able to detect significant differences in class I surface antigen expression between N2A and the G26 lines. We compared the murine neuroblastoma line Cl300 and its cloned derivative, N2A, to the lines G26-20 and G26-24. Antibody-dependent, complement-mediated cytotoxicity revealed detectable levels of both K and D region antigens on these lines. Immunocytofluorometric analysis further confirmed that these lines express high levels of class I antigens, although due to their large sizes, the surface densities of class I antigens on these cells are lower than splenocytes. This lower density of class I molecules did not impede the capacity of either the neuroblastoma or the G26 lines to serve as targets of H-2K- or D-specific T effectors. Finally, comparison of these two cell types for class I RNA transcripts also revealed no difference. Thus, our findings which are the most detailed study of these lines are drastically different from findings in humans as well as earlier findings in the murine system. Likely explanations are discussed and precautions are given for the study of class I antigen expression by these lines.