A new approach to hybrid hybridoma construction based on the use of an actinomycin D-resistant myeloma cell line

Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR E. A. Zotikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 11, pp. 511–514, November, 1991.     

Increased ELISA sensitivity using a modified method for conjugating horseradish peroxidase to monoclonal antibodies.

We investigated the importance of monoclonal antibody (MCA) purity and the input molar ratio of horseradish peroxidase (HRPO)/IgG used for MCA conjugation on various immunoenzymometric assay (IEMA) parameters. The sensitivity of IEMAs for human follicle stimulating hormone (hFSH), human chorionic gonadotropin (hCG) and the free alpha subunit of hCG (hCG alpha) could be increased up to 6-fold, whereas non-specific binding remained within tolerable limits (E less than 0.1), when MCAs purified by high performance liquid chromatography (HPLC) using a hydroxylapatite column (HPHT) were conjugated with an input molar HRPO/IgG ratio of four instead of the usual ratio of two.     

Requirement of de novo protein synthesis for aminopterin-induced apoptosis in a mouse myeloma cell line.

Cells synthesize nucleotides through de novo and salvage pathways that require the activities of dihydrofolate reductase (DHFR) and hypoxanthine-guanine phosphoribosyltransfease (HGPRT), respectively. Aminopterin, an inhibitor of dihydrofolate reductase, has been demonstrated to allow HGPRT(-) cells to be negatively selected. However, the pathway by which aminopterin leads to cell death remains to be clarified. In this study, we characterized features of cellular responses induced by aminopterin treatment in P3-X63-Ag8.653, a mouse HGPRT(-) myeloma cell line. Upon treatment with aminopterin, the cells readily underwent an apoptotic process, as assessed by DNA fragmentation assay and electron microscopic analysis. Aminopterin-induced apoptosis was drastically reduced by addition of actinomycin D and cycloheximide, indicating that active RNA and protein synthesis is required for the apoptotic effect of aminopterin. Interestingly, the induction of c-myc gene expression preceded the activity of DNA fragmentation in aminopterin-treated cells. Taken together, these results suggest that cells deficient in the salvage pathway of purine biosynthesis are susceptible to aminopterin-induced apoptosis that requires de novo synthesis of proapoptotic factors, including Myc oncoprotein.     

Thyroid microsomal/thyroid peroxidase autoantibodies show discrete patterns of cross-reactivity to myeloperoxidase, lactoperoxidase and horseradish peroxidase.

The recent cloning of the thyroid peroxidase (TPO) has shown that it is identical to the thyroid microsomal antigen (TMA), a potent antigen involved in autoimmune thyroid disease (ATD), which shares significant sequence homology with myeloperoxidase. The present study shows that autoantibodies (aAb) to the TMA/TPO antigen cross-react with human leucocyte myeloperoxidase, bovine lactoperoxidase and horseradish peroxidase. Cross-reactivity to myeloperoxidase was only apparent by ELISA using reduced and alkylated antigen preparations or by immunoblotting following denaturation with SDS. Sequential absorption of sera on SDS-denatured thyroid microsomes immobilized on Sepharose-4B followed by absorption on native microsomes removed all aAb specificities to TMA/TPO and the three peroxidase preparations, giving compelling evidence on the genuine cross-reactive nature of these aAbs. Sera from different patients contain different qualitative and quantitative specificities of aAb to the TMA/TPO antigen, confirming the polyclonal nature of this autoimmune response.     

Propagation of a mouse myeloma cell line J558L producing human CD4 immunoglobulin G1.

Transfected mouse myeloma cells are of increasing interest for the production of a wide variety of solubilised recombinant fusion proteins. A stably transfected J558L mouse myeloma subclone (J558L-CD4) secreting human CD4-immunoglobulin type G1 receptor (CD4-H gamma 1) was employed as a model system for cell suspension culture and expression of chimaeric molecules. Cells were grown up to 3-5 x 10(6) cells/ml in serum-free and protein-reduced DHI medium consisting of a mixture of DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL and Pluronic F68. Primatone RL was the essential growth-promoting factor in protein-free medium. The soluble CD4-H gamma 1 receptor, the production of which was not growth-associated, accumulated in the medium to concentrations of 40 micrograms/ml with a specific formation rate of 0.18 micrograms/10(6) cells/h in conventional cultures. The cell density was further increased by growing the cells in dialysis tubing or by using a perfusion system with cell retention. Because of the continuous exchange of nutrients and metabolic end-products average concentrations of 35 x 10(6) cells/ml were achieved. CD4-H gamma 1 accumulated in the dialysis tubing up to 1.3 mg/ml. After an initial rapid growth period, a ten-fold reduction in specific nutrient consumption rates and metabolic end-product formation was observed. Chimaeric proteins purified by protein G chromatography from conventional and perfusion cultures were indistinguishable when compared by SDS-PAGE, limited proteolysis and isoelectric focusing analysis (isoelectric point: 8.5-8.6).     

Detection of antibody-forming cells and humoral antibody to horseradish peroxidase.

Two new simple methods for detecting antibody-forming cells by hemolytic plaque assay and hemagglutinating antibody to horseradish peroxidase have been developed in mice. Both techniques utilize as target, sheep erythrocytes coupled directly with horseradish peroxidase. These assays are sensitive, antigen-specific and are useful to quantitate both direct and indirect antibody-forming cells and humoral antibodies.     

The cerebellar nucleocortical projections in the rat. A retrograde labelling study using horseradish peroxidase combined to a lectin

Following injections of wheat germ agglutinin-horseradish peroxidase conjugate in the cerebellar cortex of adult rats, retrograde cellular labelling was of two kinds within the cerebellar deep nuclei: (i) of high intensity, in those nuclear regions that also showed anterograde terminal labelling; this topographically arranged nucleocortical projection has therefore been called 'reciprocal'. 'Reciprocal' projections to zone A, to the zones C1 and C2, and to zone D come from the nucleus medialis, the interposed nuclear complex, and the nucleus lateralis, respectively; (ii) of low intensity, in nuclear areas surrounding the previous ones. No nucleocortical projections could be evidenced to either B or the C3 zones.     

The spinothalamic tract in the primate: a re-examination using wheatgerm agglutinin conjugated to horseradish peroxidase

The sites of termination of the primate spinothalamic tract have been reinvestigated using the anterograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase. Monkeys which received an injection of the conjugate at the spinal cervical level (C7-C8) displayed a "patchy" pattern of labelling in the coronal plane in the ventral posterior lateral and caudal ventrolateral nucleus. In three dimensional reconstructions this labelling appeared to be rod-like in shape. A more homogeneous pattern of labelling was present in parts of the central lateral, posterior, suprageniculate, limitans, submedius, medial dorsal, paracentral, central medial, reuniens and periventricular nucleus. Lumbar injections (L2-L3) produced a similar although less intense pattern of labelling with only the ventral posterior lateral and ventrolateral nuclei displaying an obvious topological organization. Comparison of these results with previous physiological and pharmacological reports suggests several morphological-functional correlations: first, that both the discriminative and motivational/arousal aspects of spinothalamic tract function, associated with the lateral and medial thalamic nuclei, respectively, may be conveyed by direct spinothalamic tract projections. In support of this hypothesis medial spinothalamic tract termination sites receive a homogeneous input which does not have an obvious topographical organization, whereas lateral spinothalamic tract termination sites receive a "patchy" pattern of terminals which are topographically organized; second, that the patchy pattern of labelling observed in the coronal plane in the lateral thalamus corresponds to a "rodlike" pattern of labelling in three dimensions. This "rodlike" pattern of labelling has previously been observed for medial lemniscal projections to the thalamus and has been postulated to be the thalamic equivalent of cortical "columns"; third, that there appears to be a tight overlap between spinothalamic tract terminals and opiate receptor binding in some medial but not lateral thalamic nuclei. Such an overlap may be indicative of a pharmacological difference in the types of spinothalamic tract inputs which could be modulated by opiates at the thalamic level.     

Modeling liver metastasis using a tumor cell line derived from an enhanced green fluorescent protein transgenic mouse

The liver is a common repository for metastases, second only to lymph nodes. The majority of gastrointestinal cancer deaths are attributed to liver metastasis. Researchers have widely used stable transfection of green florescent protein (GFP) to track tumor cells in the liver metastasis cascade. However, stable, sustained GFP expression in these tumor cells requires proper drug selection to avoid its loss in animal models. To overcome this, we generated a pancreatic tumor cell line that stably expressed enhanced GFP (EGFP). First, we induced a pancreatic tumor by administering 3-methylcholanthrene in the pancreas of an EGFP transgenic mouse, which had stable ubiquitous EGFP expression. Second, we established the parental pancreatic cancer cell line LG as a culture from a tumor. Third, we selected the cell line LG-L7, a highly liver-metastatic variant of LG. Both LG and LG-L7 cells exhibited a stable EGFP genotype and constant EGFP protein expression both in vitro and in vivo. Also, we could track disseminated LG cells at the single-cell level in vivo. Therefore, this novel cell model system is a useful tool for studying tumor-cell dissemination and metastasis, their underlying mechanisms, and potential therapeutic approaches for them.     

Synthesis and secretion of IgE by an established human myeloma cell line

The rate of IgE production in vitro and the cell population proliferation rate by the established human myeloma line 266 Bl have been studied quantitatively under various tissue culture conditions. The rate of extracellular IgE accumulation depended on the type of medium used, the cell density and the period of time elapsed after explantation. The maximum production rate of 8·1 × 10⁻¹² g IgE/cell/48 hr was noticed at cell densities <10⁶/30 ml and with the presence of feeder human skin fibroblasts or glia-like cells or with the use of conditioned media harvested from such cells. The rate of cell proliferation and secretion of IgE to the medium ran parallel suggesting that the IgE production is highest when cells are in the best physiological condition. During more than one year the rate of synthesis of IgE remained stable. This functionally stable human myeloma line is suitable for further studies on immunoglobulin biosynthesis at the cellular and subcellular level under the tissue culture conditions found optimal in this study.     

IFN-tau inhibits IgE production in a murine model of allergy and in an IgE-producing human myeloma cell line.

BACKGROUND: IFN-tau, a type I IFN, is an antiviral, immunomodulating, and antiproliferative agent similar to IFN-alpha and IFN-beta, but IFN-tau lacks the toxicity associated with high concentrations of these IFNs in tissue culture and in animal studies. We have previously shown that IFN-tau inhibits antibody production in a murine model of an autoimmune disease. OBJECTIVE: We investigate the effectiveness of ovine IFN-tau and other type I IFNs in suppressing the development of allergic sensitization in a murine model of allergy by using ovalbumin (OVA) antigen as an allergen and in suppressing IgE production by using a human IgE-producing myeloma cell line.Methods and Results: Mice that were treated with IFN-tau in vivo before and after intraperitoneal immunization with aluminum hydroxide-precipitated OVA had significantly lower OVA-specific IgE levels than the PBS-treated group. IFN-tau-treated mice had reduced inflammatory cell infiltration into the lung tissue. Furthermore, in vitro IFN-tau treatment of splenocytes taken from OVA-immunized mice suppressed OVA-induced proliferation. Also, treatment of the IgE-producing human myeloma cell line U266BL with IFN-tau-reduced IgE production and inhibited cell proliferation compared with media controls. Similar suppression of proliferation and inhibition of IgE production was seen with other type I IFNs, as well as a humanized IFN-tau/IFN-alphaD chimeric that consists of residues 1 to 27 of the ovine IFN-tau and residues 28 to 166 of the human IFN-alphaD. The chimeric was not toxic to human peripheral white blood cells at concentrations as high as 10(5) U/mL, whereas human IFN-alphaD was toxic at 10(3) U/mL. CONCLUSION: These data suggest that IFNs may be useful in preventing allergic sensitization by suppressing the production of allergen-specific IgE antibodies without toxic side effects.     

Electron microscopic study on permeability of coronary artery wall of normotensive rabbits using horseradish peroxidase as a tracer.

In order to study the pathways in the coronary artery vessel wall horseradish peroxidase (HRP) was used as a tracer. HRP was injected directly in the left ventricle of rabbit hearts. 30 seconds after intracardial injection of HRP in normotensive rabbits, peroxidase reaction products could be located in random areas of the coronary arterial system and could then be observed in all three layers of the coronary walls at these locations. HRP reaches the subendothelial space of the coronary arteries along two pathways: through intercellular junctions and by plasmalemmal vesicles. Through the fenestations of the internal elastic lamina HRP reaches the media and can be detected between the media smooth muscle cells. The influence of the methodology on the results is discussed.     

Examination of neurons in wild type and mutants of Caenorhabditis elegans using antibodies to horseradish peroxidase

Antibodies to horseradish peroxidase (HRP) recognize 27 of 302 neurons and several non-neuronal cells in adult hermaphrodites of the soil nematode Caenorhabditis elegans and can be used to label these cells for cytological analysis in whole animals. The antibodies bind to the anterior members, but not to the posterior members of a set of mechanosensory neurons in wild type animals. Binding to one of the posterior mechanosensory neurons (PVM) occurs when this neuron migrates to an abnormal anterior position in mab-5 mutant animals, suggesting that expression of the epitope recognized by these antibodies is position dependent or that mab-5 mutations transform PVM into AVM intrinsically. The antibodies were used to characterize morphologies of two pairs of lumbar neurons (PHC and PVN) in uncoordinated mutants representing 95 unc genes. PHC and PVN morphologies were normal in most of the unc mutants examined, however, in mutants of 9 unc genes (unc-6, unc-13, unc-33, unc-44, unc-51, unc-61, unc-71, unc-73, and unc-98), misdirected PHC and/or PVN processes were observed at a high frequency. The morphologies of 2 other lumbar neurons, PHA and PHB, were determined previously in these mutants (Hedgecock et al., 1985). Mutations in most, but not all of these 9 unc genes affect the growth of the embryonic lumbar neurons PHA and PHB differently than they affect the growth of the postembryonic lumbar neurons PHC and PVN, indicating that these neurons require different, but overlapping sets of genes for different stages of normal growth and guidance.     

Examination of neurons in wild type and mutants of Caenorhabditis elegans using antibodies to horseradish peroxidase

Antibodies to horseradish peroxidase (HRP) recognize 27 of 302 neurons and several non-neuronal cells in adult hermaphrodites of the soil nematode Caenorhabditis elegans and can be used to label these cells for cytological analysis in whole animals. The antibodies bind to the anterior members, but not to the posterior members of a set of mechanosensory neurons in wild type animals. Binding to one of the posterior mechanosensory neurons (PVM) occurs when this neuron migrates to an abnormal anterior position in mab-5 mutant animals, suggesting that expression of the epitope recognized by these antibodies is position dependent or that mab-5 mutations transform PVM into AVM intrinsically. The antibodies were used to characterize morphologies of two pairs of lumbar neurons (PHC and PVN) in uncoordinated mutants representing 95 unc genes. PHC and PVN morphologies were normal in most of the unc mutants examined, however, in mutants of 9 unc genes (unc-6, unc-13, unc-33, unc-44, unc-51, unc-61, unc-71, unc-73, and unc-98), misdirected PHC and/or PVN processes were observed at a high frequency. The morphologies of 2 other lumbar neurons, PHA and PHB, were determined previously in these mutants (Hedgecock et al., 1985). Mutations in most, but not all of these 9 unc genes affect the growth of the embryonic lumbar neurons PHA and PHB differently than they affect the growth of the postembryonic lumbar neurons PHC and PVN, indicating that these neurons require different, but overlapping sets of genes for different stages of normal growth and guidance.     

A re-examination of the rubro-olivary tract in the cat,using horseradish peroxidase as a retrograde and an anterograde neuronal tracer

Most authors using free horseradish peroxidase as a retrograde tracer have had difficulties in demonstrating a rubro-olivary tract. Injections of this tracer into the inferior olive in animals of different species resulted in only a few retrogradely labelled rubral cells within the rostral part of the ipsilateral nucleus. The present study, which is based on the highly sensitive technique of Mesulam (1978), confirms this observation. Injections of free horseradish peroxidase (Serva or Sigma VI type) into the dorsal lamella of the principal olive in eleven cats gave a positive retrograde labelling of rostral rubral cells in only three animals, with a very few cells in each case. In contrast to this, olivary injections of horseradish peroxidase labelled lectin (wheat germ agglutinin) result in an abundance of retrogradely labelled cells within the rostral part of the ipsilateral red nucleus, the great majority of these cells being of the small and medium-sized type. Many of the retrogradely labelled rubral cells are only faintly stained. However, free horseradish peroxidase appears to be well suited as an anterograde tracer for a demonstration of the rubro-olivary tract.The findings are discussed in relation to the observation that in the cat only peroxidase conjugated to lectin can be used successfully as a retrograde tracer for a visualization of the rubro-olivary connection.     

Projections to the ventral surface of the cat brainstem demonstrated by horseradish peroxidase.

Topical application of horseradish peroxidase (HRP) to the ventral surface of the medulla oblongata revealed various cells with axons terminating near this region. Fibers traced from the ventral surface originated in the nucleus of the tractus solitarius and the dorsal nucleus of the vagus, both near the dorsal surface of the medulla. Projections from the cochlear nucleus were also observed. Using short HRP contact time, fibers were shown which terminate at, or near, the ventral surface. The possible involvement of these projections in various responses mediated from the ventral surface is discussed.