Electrical excitability and chemosensitivity of mouse neuroblastoma X mouse or human fibroblast hybrids

Intracellular microelectrode recording techniques were used to measure passive membrane properties, electrical excitability and chemosensitivity of mouse neuroblastoma cells and somatic cell hybrids formed between these cells and either L cells or human diploid fibroblasts. Different clones of the hybrid cells showed varying degrees of neuronal or fibroblastic membrane-differentiated function; a selection technique involving incubation of the cells with aminopterin gave quite homogeneous non-dividing populations of cells within a given clone of the neuroblastoma x L cell hybrids. Despite relatively uniform chromosomal numbers within a given clone, the neuroblastoma x human fibroblast hybrids were morphologically and electrophysiologically heterogeneous. The possibility is considered that this may represent the effect of variable segregation of the human chromosomal complement.     

Expression of the neuron-specific protein, 14-3-2, and steroid sulfatase in neuroblastoma cell hybrids

Three series of neuroblastoma X fibroblast hybrid clones were isolated from crosses between mouse or human fibroblasts and mouse or human neuroblastoma cell lines by virus-mediated cell fusion. The expression of 14-3-2 protein (an acidic protein specific to neurons) and steroid sulfatase activity was studied in parental and hybrid cell lines. Steroid sulfatase was extinguished in hybrids when only one parent expressed the enzyme, but was expressed in one hybrid combination in which both parents expressed the enzyme. The neuron-specific 14-3-2 protein, on the other hand, continued to be expressed in all three series of neuroblastoma x fibroblast hybrids. In most cases where these pheno-types were expressed, they also exhibited temporal modulation; that is, specific activity is low during logarithmic growth and increases markedly during stationary phase. The glial-specific protein S-100 is absent from all parents and hybrids. The results are discussed in terms of mechanisms of regulation of differentiated phenotypes in mammalian cells.     

Membrane excitability expressed in human neuroblastoma cell hybrids

The voltage-sensitive Na+ channel is responsible for the action potential of membrane electrical excitability in neuronal tissue. Three methods were used to demonstrate the presence of neurotoxin-responsive Na+ channels in two hybrid cell lines resulting from the fusion of excitable human neuroblastoma cells with mouse fibroblasts. Only one of the two electrically active hybrid cell lines maintained the sensitivity of the neuroblastoma parent to tetrodotoxin (TTX). The other hybrid, although electrically active, was not responsive to TTX or scorpion venom. Comparisons of the patterns of expression of membrane excitability and of chromosome complements in these human neuroblastoma cell hybrids suggest that the phenotype of membrane excitability is composed of genetically distinct elements.     

High activity of choline acetyltransferase induced in neuroblastoma X glia hybrid cells

Hybrids were made between rat glioma X mouse neuroblastoma cell lines and were examined for the specific activities of choline acetyltransferase and acetylcholinesterase. The specific activities of choline acetyltransferase of the hybrids were as high as those in normal brain, whereas neither parent line showed appreciable activities. The electrical excitability of the neuroblastoma cells was retained in the hybrids.     

Excitability and chemosensitivity properties of a somatic cell hybrid between mouse neuroblastoma and sympathetic ganglion cells

A somatic cell hybrid line, NX-31, formed by fusion of mouse neuroblastoma and mouse sympathetic ganglion cells has been studied for its electrophysiologic properties and its chemosensitivity to iontophoretically applied acetylcholine (ACh). The cell lines (parent and hybrid) were treated and studied in media containing mM dibutyryl cyclic AMP (db-cAMP) in order to obtain them in a maximally differentiated state. The hybrid cell line was electrically excitable and its excitability characteristics were found to be closer to those of its putative neuronal parent than were those of its neuroblastoma parent. In particular, increases were observed in the percentage of cells giving action potentials when electrically stimulated at their resting membrane potentials, in the maximal rate of rise (dV/dt) of the elicited spikes, and in the percentage of cells displaying repetitive-firing ability. Fifty percent of the hybrid cells were sensitive to ACh. Two types of depolarizing responses were evoked—an early, rapidly developing response and, less frequently, a late, slowly developing response. The first type of depolarization appears to be mediated by a nicotinic cholinergic receptor while the latter type does not. Neuroblastoma cells may exhibit only the early, rapid response; sympathetic neurons show depolarizations corresponding to both types of responses. These findings suggest that the phenotypes expressed by NX-31 hybrid cells more closely resemble those of sympathetic neurons than do those of their neuroblastoma parents. The possibility is raised of further exploiting the strategy of hybridizing a dividing cell line with other types of neurons to obtain new neuronal cell lines.     

Expression and extinction of glial properties in glioma X neuroblastoma cell hybrids

Rat glioma cells (clone C₆TK⁻) were hybridized with mouse neuroblastoma cells (clone NA), and 18 primary and secondary hybrid clones containing one chromosome set from each parent were isolated. The hybrids were assayed for the glial marker enzymes 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycerol-3-phosphate dehydrogenase (GPDH). In many of the hybrid clones, the levels of CNP and GPDH were reduced to 5–20% of the activity of C₆TK⁻, as has been observed in other classes of glial X non-glial cell hybrids. In some hybrid clones, however, GPDH and CNP were expressed at high activity. Rat (glial) GPDH activity was not reduced in these clones, but mouse GPDH activity remained low, and was not “de-repressed” or “activated”. This suggests that the controls governing differentiation in neuroblastoma cells and extinction in hybrids may differ in some important details. There was a strong positive correlation between the specific activities of CNP and GPDH in the hybrid clones, suggesting that a mechanism regulates the activity of these two glial enzymes coordinately.     

Analysis of myogenesis by somatic cell hybridization. II. Retention of myogenic competence and suppression of transformed properties in hybrids between differentiation competent and incompetent rat L6 myoblasts

This study describes the characteristics of hybrids between two closely related rat myoblast lines, which differ both in the ability to express their program of differentiation and in the expression of neoplastic properties. Myogenic, nonneoplastic L6J1-S cells were hybridized with nonmyogenic, neoplastic L6J1-N1 cells. Six hybrid clones were isolated and expanded for analysis of myogenic competence, and four of these clones were also evaluated for parameters of transformation, including tumorigenicity, ability to clone in agar, and surface fibronectin. In addition to our analysis of isolated clones, we also assessed myogenic differentiation in colonies representing 226 early hybrid clones. Results of all these analyses demonstrate that the myogenic phenotype is retained and that the tumorigenic/transformed phenotype is suppressed in the hybrids. Furthermore, our results indicate that when the programs for myogenesis and neoplastic transformation are confronted within a single cell, they are expressed as mutually exclusive alternatives. In contrast to these results on myogenic X nonmyogenic L6 hybrids, it has been reported that isolated clones of A9 X L6 exhibited extinction of myogenic competence and retention of transformed properties. We have evaluated myotube formation in over 300 early hybrid clones between A9 and either diploid or subtetraploid L8 rat myoblasts. Our results demonstrate that all of these hybrid clones exhibit extinction regardless of the ploidy of the myoblast parent, and they further indicate that extinction is not a consequence of chromosome loss. These results support the conclusion that in A9 X L6 hybrids, the nonmyogenic, transformed phenotype is dominant.     

Further evidence that muscarinic cholinergic receptors of 1321N1 astrocytoma cells couple to a guanine nucleotide regulatory protein that is not Ni.

Inhibitory coupling of receptors to adenylate cyclase previously has been shown to be relatively sensitive to inactivation by alkylation with N-ethylmaleimide (NEM). Modification of the inhibitory guanine nucleotide regulatory protein, Ni, has been proposed to be responsible for this effect. The effects of NEM on GTP-sensitive binding of carbachol to muscarinic cholinergic receptors has been compared in a cell line (1321N1 human astrocytoma cells) in which these receptors stimulate phosphoinositide breakdown and in a cell line (NG108-15 neuroblastoma X glioma cells) in which activation of these receptors results in inhibition of adenylate cyclase. Pretreatment of membrane preparations from 1321N1 cells with NEM resulted in a concentration-dependent decrease in the extent of pertussis toxin-catalysed [32P]ADP-ribosylation of a 41 000 Da protein previously proposed to be the alpha subunit of Ni. Under conditions where 32P-labelling of Ni in 1321N1 membranes was reduced by NEM by 90%, no effect was observed on the extent of guanine nucleotide-sensitive high-affinity binding of carbachol to muscarinic cholinergic receptors. In contrast, treatment of NG108-15 membranes with NEM under the same conditions resulted in complete loss of high-affinity guanine nucleotide sensitive binding of carbachol. These results illustrate another difference between the muscarinic receptor population of these two cell lines, and support the previous proposal that muscarinic receptors of 1321N1 cells couple to a guanine nucleotide regulatory protein that is not Ni.     

Glucocorticoid responsiveness of hepatoma cell hybrids

The dominance or recessiveness of glucocorticoid responsiveness and albumin production of different hepatoma cell lines were investigated using somatic cell hybrids. The majority of the intraspecific, intraorgan hybrids between glucocorticoid sensitive, tyrosine aminotransferase (TAT) non-inducible, albumin non-secreting parents and glucocorticoid resistant, TAT-inducible, albumin secreting parents were glucocorticoid sensitive, TAT non-inducible and albumin non-producing. The TAT inducibility was extinguished in interspecific, interorgan hybrids of TAT-inducible hepatoma and non-inducible Chinese hamster fibroblast parents. No TAT reexpression was observed among the many independent hybrid clones. The experiments provided evidence for the non-coordinate control of the expression of differentiated functions in hepatoma hybrid cells.     

Preferential generation of human T cell hybrids with a tetraploid fusion partner

The factors determining successful derivation of human T lymphocyte hybrids are largely unknown. This report describes diploid and tetraploid clones of the T cell line CEM which were fused with either a human T cell line (Jurkat) or with peripheral blood lymphocytes (PBL). Fusions of all CEMR clones with the Jurkat cell line yielded hybrids at a very high frequency (1 X 10(-4)). Fusion of diploid clones of CEM with PBL yielded no hybrids, whereas fusion of tetraploid clones of CEM with PBL resulted in growth frequencies of 1 to 3 X 10(-6). Enumeration of hybrids immediately after fusion indicated that in all cases, fused cells represented 5 to 10% of the population. That the ability to yield viable hybrids after fusion was a characteristic of tetraploid cells was indicated by the finding that tetraploid variants of a diploid clone could also yield viable hybrids after fusion. Possible mechanisms for the difference in results generated with diploid and tetraploid cells, and characteristics of the hybrid cells generated, are also discussed.     

乙酰胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchR1表达的影响

用CCK-8比色法和流式细胞术,检测乙酰胆碱（acetylcholine,Ach）及拮抗剂阿托品（atropine,Atro）对SK-N-SH细胞增殖活性和周期分布的调节作用;进一步用荧光定量PCR、免疫印迹和流式细胞间接免疫荧光技术,分析SK-N-SH细胞毒蕈碱受体亚型Ⅰ型（mAchR1）和c-fos的表达差异。结果表明,1mmol／L Ach对SK-N-SH细胞有明显促增殖作用,而1mmoL／L Atro阻滞细胞从S期向G2／M期移行;1mmol／L Ach与1mmol／L Atro均反馈调节mAchR1的蛋白水平。但mAchR1 mRNA的表达不受影响;1mmol／L Ach显著上调c-fos mRNA和Fos蛋白的表达,但这种作用可被Atro逆转。提示胆碱类受体参与配基对肿瘤细胞的促增殖作用。     

乙酰胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchRl表达的影响

用CCK-8比色法和流式细胞术,检测乙酰胆碱(acetylcholine,Ach)及拮抗剂阿托品(at-ropine,Atro)对SK-N-SH细胞增殖活性和周期分布的调节作用;进一步用荧光定量PCR、免疫印迹和流式细胞间接免疫荧光技术,分析SK-N-SH细胞毒蕈碱受体亚型Ⅰ型(mAchR1)和c-fos的表达差异。结果表明,1mmol/LAch对SK-N-SH细胞有明显促增殖作用,而1mmol/LAtro阻滞细胞从S期向G_2/M期移行;1mmol/LAch与1mmol/LAtro均反馈调节mAchR1的蛋白水平,但mAchR1mRNA的表达不受影响;1mmol/LAch显著上调c-fosmRNA和Fos蛋白的表达,但这种作用可被Atro逆转。提示胆碱类受体参与配基对肿瘤细胞的促增殖作用。     

Somatic cell hybridization of mouse myeloma cells

Somatic cell hybrids between different mouse myeloma cell lines have been readily isolated using modifications of existing techniques. The hybrid nature of these cells was established by HAT or HAT-ouabain selective procedures, their chromosome number, and, in one case, H-2 surface antigen expression. Three hybrid cell lines are described here in detail: an IgG_2b, ? X IgG_2a, ?; an IgG₁, ? X IgG_2b, ?; and an IgG₁, ? X IgM, λ. In all cases, both parental types of H and L chains are expressed in the hybrid cells and no new chains are observed. However, molecules possessing disulfide-bonded mixtures of parental H and/or L chains are seen. Analysis of subclones of these hybrids indicates considerable stability in the expression of the immunoglobulins for up to 13 months. However, segregant clones no longer synthesizing one or more of the parental H or L chains arise frequently.     

Expression of genes for metabolism of cyclic adenosine 3':5'-monophosphate in somatic cells. beta-Adrenergic and PGE1 receptors in parental and hybrid cells.

Using the ligands [125I]iodohydroxybenzylpindolol and [3H]prostaglandin E1 ([3H]PGE1), we have studied the relationship of receptors for beta-adrenergic agents and for PGE1 to adenylate cyclase in membranes of parental, hybrid, and variant mammalian cell lines. Fusion of parental clones responsive to beta-adrenergic agonists (beta+) with unresponsive clones (beta-) produced hybrid clones with a greatly diminished beta-adrenergic response; beta+ X beta leads to beta-. Binding studies with [125I]iodohydroxybenzylpindolol showed a decreased concentration of beta receptors in six such hybrid clones. Thus, paucity of beta-adrenergic receptors is probably a sufficient, albeit not necessarily complete, explanation for the decreased beta-adrenergic responsiveness of the hybrid clones. When a clone with beta receptor but without apparent adenylate cyclase activity (HC-1) was hybridized with a beta- clone that has adenylate cyclase (B82), a responsive hybrid clone was obtained. In nine cell hybrids produced by the fusion of clones responsive (PGE1+) and unresponsive (PGE1-) to PGE1, high affinity binding sites for [3H]PGE1 were expressed in the same manner as was PGE1-sensitive adenylate cyclase: PGE1+ X PGE1 leads to PGE1+. The chemical specificities and affinities of the parental receptors and responsive adenylate cyclases were faithfully reproduced in the hybrid clones. Activation by PGE1 was proportional to the occupation of the high affinity receptors. In a wild type lymphoma clone (24.3.2), the concentration dependences for binding of [3H]PGE1 and for activation of adenyalte cyclase by PGE1 were identical. In a variant lymphoma clone (94.15.1) lacking adenylate cyclase activity, no high affinity receptors for PGE1 were detected, whereas beta-adrenergic receptors have been demonstrated in this variant clone (Insel, P.A., Maguire, M.E., Gilman, A.G., Coffino, P., Bourne, H., and Melmon, K. (1976) Mol. Pharmacol. 12, 1062-1069). Hybrid cells formed by the fusion of 94.15.1 with cell line RAG (PGE1-) responded to PGE1. Clone 94.15.1 may have receptors for PGE1 of reduced affinity or in low concentration. Alternatively, RAG and 94.15.1 may have complementary genetic defects such that the RAG X 94.15.1 hybrid cells express a hormonally responsive receptor-adenylate cyclase system.     

Analysis of myogenesis by somatic cell hybridization: III. Myogenic competence of hybrids derived from rat L6 myoblasts and mouse primary fibroblasts and myoblasts

Hybrid cells derived from rat L6 myoblasts and mouse primary fibroblasts (M x F hybrids), as well as those derived from rat L6 myoblasts and mouse primary myoblasts (M x M hybrids), were examined for their ability to engage in myogenesis as judged by muscle fiber formation plus the expression of skeletal muscle myosin and creatine kinase (CK). Of 172 primary hybrid colonies scored, 59% were myogenic in the M x F fusion and 97% exhibited muscle fiber formation in the M x M fusion. Individual hybrid clones from each cross were isolated, expanded and analyzed for myogenic capabilities as well. All three M x M and all ten M x F isolated clones exhibited preferential elimination of mouse chromosomes. Nonetheless, all were capable of fusing spontaneously and of elaborating skeletal muscle myosin and CK. The three M x M hybrids expressed only MM-CK whereas nine out of ten M x F hybrids produced all three CK isoenzymes (MM, MB, BB). These results suggest that M X M hybrids express CK patterns reminiscent of the rat L6 parental cells while M X F hybrids apparently mimic mouse muscle fiber CK patterns. Various models are discussed which address these phenomena.     

Influence of Zinnia angustifolia HBK genotype on embryonic and vegetative development of Z. angustifolia x Z. elegans Jacq. interspecific hybrids

Summary Interspecific crosses between Zinnia angustifolia clones (maternal parents) and Z. elegans lines (paternal parents) were performed to investigate postzygotic barriers among Z. angustifolia X Z. elegans hybrids and to determine influence of parental genotype on embryonic and vegetative development of interspecific hybrids. Variation in percentage of emerged seedlings (PES) and percentage of morphologically normal hybrids (PNH) was attributable to Z. angustifolia clones with minor or no effect attributable to Z. elegans lines. Heterogeneity in PES values among Z. angustifolia clones was due to differences in amount of hybrid embryo breakdown and ungerminable seed. Cytological observations of normal and abnormal interspecific hybrids revealed similar chromosome numbers (2n=23) but indicated a low mitotic index for abnormal hybrids. Genetic analysis of PES and PNH suggested control by multiple genes inherited from the Z. angustifolia genome. Adequate sampling of the Z. angustifolia gene pool would permit exploitation of genetic variability present within the species and allow improvements in PES and PNH for interspecific hybrids.Scientific Article No. A-4479, Contribution No. 7472 of the Maryland Agricultural Experiment Station, Department of Horticulture     

Expression of A and B types of monoamine oxidase in neuroblastoma hybrid cells

Monoamine oxidase (MAO) activity towards kynuramine as substrate was measured in 6 hybrid cells derived by fusion of neuroblastoma and glioma, liver or brain cells, and was compared with that of parental or non-parental clones. Activities varied from the lowest level of less than 0.15 pmol/min/mg protein in a neuroblastoma clone NB2A to the highest level of 127 pmol/min/mg protein in NCB20 mouse neuroblastoma × Chinese hamster embryo brain hybrid cells. The relative proportions of A and B types of MAO activities were determined in homogenates of each cell line by inhibition curves with clorgyline and deprenyl. Although the A type activity was found in all cell lines measured, MAO A was predominant in 9 clones, except for NCB20 hybrid cells, N4G-B-a neuroblastoma × glioma hybrid cells, and G8-1 myoblast. The ratio of type A/type B activity in NCB20, N4G-B-a and G8-1 cells was 20/80, 75/25 and 95/5, respectively. The results suggest that NCB20 cells are highly enriched in MAO type B, and that the NCB20 cell is an excellent model for studying the type B activity found in the brain in vivo.     

Differences in the functional responses of two cell lines each expressing Pi-hydrolysis-coupled muscarinic receptors

Fluorescent oxonol dyes were used to measure changes in the membrane potential of two different cell lines each expressing Pi-hydrolysis coupled muscarinic receptors. Both SK-N-SH human neuroblastoma cells and m1-transfected A9 L cells express muscarinic receptors, which, when stimulated, elicit a large increase in intracellular calcium, and release of inositol phosphates. Despite the similarity in this second-messenger response, muscarinic stimulation resulted in a hyperpolarization in the transfected A9 L cells whereas a small depolarization was observed in the neuroblastoma cells. The carbachol-mediated hyperpolarization of the transfected A9 L cells could be mimicked by increasing intracellular calcium with the ionophore A23187, suggesting, that it may be mediated by calcium-activated potassium channels. Exposure of SK-N-SH cells to A23187, on the other hand, had no effect on the membrane potential. These studies demonstrate that the activation of a second messenger system does not solely dictate the electrophysiological response of a cell, but that other factors such as the expression of ion-channels is critical in the determination of that response.     

Expression of nervous system antigen-3 by lines of mouse fibroblasts and kidney cells and continued expression in hybrid cells

In a survey of the expression on cultured mouse cells of the cell surface antigen known as nervous system antigen-3 (NS-3), it was found that RAG, a renal adenocarcinoma line, expressed that antigen. It was also observed that 3T3, a fibroblast line of unknown tissue origin, expressed NS-3. Cells of these two lines were hybridized with cells of two mouse L cell lines that did not express NS-3. Four hybrid clones were tested for both the 3T3 × L cell cross and the RAG × L cell cross, and all the hybrids were found to be NS-3 positive. All the hybrids had at least 40% as much activity as the NS-3 positive parent. Of the four parental mouse cell lines used, only 3T3 expressed Thy-1.2 antigen on the cell surface. In contrast to the continued expression of NS-3 on hybrid cells, Thy-1.2 antigen was not detectable on two clones of 3T3 × L cell hybrids that were tested.     

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.