Showing papers on "Cell culture published in 1981"

Establishment in culture of pluripotential cells from mouse embryos

Abstract: Pluripotential cells are present in a mouse embryo until at least an early post-implantation stage, as shown by their ability to take part hi the formation of chimaeric animals1 and to form teratocarcinomas2. Until now it has not been possible to establish progressively growing cultures of these cells in vitro, and cell lines have only been obtained after teratocarcinoma formation in vivo. We report here the establishment in tissue culture of pluripotent cell lines which have been isolated directly from in vitro cultures of mouse blastocysts. These cells are able to differentiate either in vitro or after innoculation into a mouse as a tumour in vivo. They have a normal karyotype.

F4/80, a monoclonal antibody directed specifically against the mouse macrophage

Abstract: A hybridoma clone which secretes a macrophage (MΦ)-specific monoclonal antibody, F4/80, was produced by fusing spleen cells from a rat hyperimmunized with cultured thioglycollate-induced mouse peritoneal MΦ with a mouse myeloma, NS1. Binding of antibody to primary cells and cell lines was detected by radioimmune indirect binding assay, autoradiography or fluorescence-activated cell sorter analysis. F4/80 binds to mouse MΦ from the peritoneal cavity or other sources, blood monocytes, MΦ derived from bone marrow precursors in culture and MΦ-like cell lines, but not to other cells, including polymorphonuclear leukocytes, lymphocytes or fibroblasts. F4/80 does not bind to MΦ via Fc receptors, is not cytotoxic and is of the rat IgG2b subclass. Since F4/80 binds to all MΦ defined by adherence, morphology and immune phagocytosis, it provides a new marker to define the MΦ in the mouse. Large differences in expression of antigen F4/80 were found, depending on intraperitoneal stimulation, time in culture and stage of maturation. Immunoprecipitation experiments demonstrated that the antigen F4/80 is part of a component of Mr 160000 which is synthesized by the MΦ and, at least in part, exposed on the cell surface.

T cell growth factor receptors. Quantitation, specificity, and biological relevance

Abstract: To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain at 37 degrees C) and was both saturable and largely reversible. In addition, at 37 degrees C, a receptor- and lysosome-dependent degradation of TCGF occurred. Radiolabeled TCGF binding was specific for activated, TCGF-responsive T cells. Whereas unstimulated lymphocytes of human or murine origin and lipopolysaccharide-activated B cell blasts expressed few if any detectable binding sites, lectin- or alloantigen-activated cells had easily detectable binding sites. Moreover, compared with lectin- or alloantigen-activated T cells, long-term TCGF-dependent cytolytic and helper T cell lines and TCGF-dependent neo-plastic T cell lines bound TCGF with a similar affinity (dissociation constant of 5-25 pM) and expressed a similar number of receptor sites per cell (5,000-15,000). In contrast, a number of TCGF-independent cell lines of T cell, B cell, or myeloid origin did not bind detectable quantities of radiolabeled TCGF. Binding of radiolabeled TCGF to TCGF-responsive cells was specific, in that among several growth factors and polypeptide hormones tested, only TCGF competed for binding. Finally, the relative magnitude of T cell proliferation induced by a given concentration of TCGF closely paralleled the fraction of occupied receptor sites. As the extent of T cell clonal expansion depends on TCGF and on the TCGF receptor, the dissection of the molecular events surrounding the interaction of TCGF and its receptor that these studies permit, should provide new insight into the hormonelike regulation of the immune response by this lymphokine.

Effects of sodium butyrate, a new pharmacological agent, on cells in culture

Abstract: Sodium butyrate, at millimolar concentrations, when added to cell cultures produces many morphological and biochemical modifications in a reversible manner. Some of them occur in all cell lines. They concern regulatory mechanisms of gene expression and cell growth: an hyperacetylation of histone resulting from an inhibition of histone deacetylase and an arrest of cell proliferation are almost constantly observed. Some other modifications vary from one cell type to another: induction of proteins, including enzymes, hormones, hemoglobin, inhibition of cell differentiation, reversion of transformed characteristics of cells to normal morphological and biochemical pattern, increase in interferon antiviral efficiency and induction of integrated viruses. Most if not all these effects of butyrate could result from histone hyperacetylation, from changes in chromatin structures as measured by accessibility to DNases and from modifications in cytoskeleton assembly. We do not know at the present time whether butyrate acts on a very specific target site in cell or if it acts on several cell components.

Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin.

Abstract: A cell surface glycoprotein antigen with an apparent molecular weight of about 100,000 that is selectively expressed on proliferating cells was purified from deoxycholate-solubilized membranes of a cultured human leukemic thymus-derived (T) cell line by affinity chromatography on a monoclonal antibody-Sepharose column. A conventional xenoantiserum prepared by immunization with the affinity-purified glycoprotein was found to contain antibodies against a serum component that bound tightly to cultured cells. This molecule was shown to be specifically associated with the cell surface glycoprotein purified by immunoprecipitation from lysates of cells. We have identified the serum component as transferrin and conclude that the membrane glycoprotein is the cell surface transferrin receptor.

Heterogeneity of Malignant Cells from a Human Colonic Carcinoma

Abstract: Three subpopulations of malignant cells were isolated from a primary cell culture of a single human colonic carcinoma. The variant cells were established as cell lines designated HCT 116, HCT 116a, and HCT 116b, respectively. In vitro characterizations of the variant lines included growth in 0.5% agarose and growth on confluent layers of mouse fibroblasts. HCT 116a showed the highest colony formation in agarose and on confluent fibroblasts, while colony formation by HCT 116 was higher than that of HCT 116b in both of these systems. All of the variant lines were tumorigenic in athymic nude mice given injections of 10 x 10(6) cells, but the time between inoculation and tumor development (latency period) was approximately 10 times longer for HCT 116b as for HCT 116a and 8 times longer than for HCT 116. HCT 116b was not tumorigenic at an inoculum of 5 x 10(6) cells, while both HCT 116 and 116a were tumorgenic at this level. However, HCT 116a was clearly more tumorigenic than was HCT 116 on the basis of the number of animals developing tumors at inoculate of both 10 x 10(6) and 5 x 10(6) cells and on the basis of their differences in latency periods. While all the cell lines had near diploid numbers of chromosomes, each line showed a distinct histological pattern when grown as xenografts in athymic nude mice.

Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells.

Abstract: The 54K cellular tumor antigen has been translated in vitro, using messenger ribonucleic acids from simian virus 40 (SV40)-transformed cells or 3T3 cells. The in vitro 54K product could be immunoprecipitated with SV40 tumor serum and had a peptide map that was similar, but not identical, to the in vivo product. The levels of this 54K protein in SV3T3 cells were significantly higher than those detected in 3T3 cells (D. I. H. Linzer, W. Maltzman, and A. J. Levine, Virology 98:308-318, 1979). In spite of this, the levels of translatable 54K messenger ribonucleic acid from 3T3 and SV3T3 cells were roughly equivalent or often greater in 3T3 cells. Pulse-chase experiments with the 54K protein from 3T3 or SV3T3 cells demonstrated that this protein, once synthesized, was rapidly degraded in 3T3 cells but was extremely stable in SV3T3 cells. Similarly, in an SV40 tsA-transformed cell line, temperature sensitive for the SV40 T-antigen, the 54K protein was rapidly turned over at the nonpermissive temperature and stable at the permissive temperature, whereas the levels of translatable 54K messenger ribonucleic acid at each temperature were roughly equal. These results demonstrate a post-translational regulation of the 54K cellular tumor antigen and suggest that this control is mediated by the SV40 large T-antigen.

Metastatic Potential is Positively Correlated with Cell Surface Sialylation of Cultured Murine Tumor Cell Lines

Abstract: The ability of murine tumor cells to metastasize spontaneously from subcutaneous sites is positively correlated with the total sialic acid content of the cells in culture, the degree to which the sialic acid is exposed on the tumor cell surface, and, most strongly, with the degree of sialylation of galactosyl and N-acetylgalactosaminyl residues in cell surface glycoconjugates. These findings suggest that sialic acid on the cell surface may play a role in tumor cell metastasis.

Epidermal cell (keratinocyte)-derived thymocyte-activating factor (ETAF).

Abstract: In order to determine whether keratinocytes play a role in the modulation of the immune response, we investigated the murine keratinocyte cell line Pam 212. In culture these cells generate a substance with a biologic activity that greatly enhances phytohemagglutinin-induced thymocyte proliferation. We have, therefore, called this substance epidermal cell thymocyte-activating factor (ETAF). This keratinocyte-derived supernatant activity is mainly produced at the onset of the logarithmic growth phase and is directly mitogenic for murine thymocytes. Although ETAF by itself exhibits no T cell growth factor activity, ETAF enhances Interleukin 2 production by mitogen-stimulated murine spleen cells. Murine ETAF is not genetically restricted and lacks species specificity since it decreases lectin-induced proliferation of human peripheral blood lymphocytes (as well as murine spleen cells) and also enhances the production of human Interleukin 2. The factor has a m.w. between 15,000 and 25,000 as determined by gel filtration and elutes as a single peak from anion exchange chromatography columns. The activity is maintained mainly at alkaline pH and is rapidly destroyed at temperatures above 60 degrees C. These observations suggest that epidermal cells may interact with the immune system by elaborating nonspecific factors that modulate lymphocyte proliferation and augment lymphokine production.

Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells

Abstract: A clonal hamster beta cell line (HIT) was established by simian virus 40 transformation of Syrian hamster pancreatic islet cells. Cytoplasmic insulin was detected in all cells by indirect fluorescent antibody staining, and membrane-bound secretory granules were observed ultrastructurally. Acidified-ethanol extracts of HIT cell cultures contained hamster insulin as determined by radioimmunoassay, radioreceptor assay, and bioassay. One subclone at passage 39 contained 2.6 micrograms of insulin per mg of cell protein. [3H]Leucine-labeled HIT insulin and proinsulin were identical to islet-derived proteins when compared by NaDodSO4/polyacrylamide gel electrophoresis of immunoprecipitates. HIT cell insulin secretion was stimulated by glucose, glucagon, and 3-isobutyl-1-methylxanthine. Insulin secretion at optimal glucose concentration (7.5 mM) was 2.4 milliunits per 10(6) cells per hr. Somatostatin and dexamethasone markedly inhibited HIT insulin secretion. The HIT cell line represents a unique in vitro system for studying beta cell metabolism and insulin biosynthesis.

A monoclonal antibody (BA-1) reactive with cells of human B lymphocyte lineage.

Abstract: NALM-6-M1 is an acute lymphoblastic leukemia cell line previously shown in our laboratory to express the pre-B lymphocyte phenotype, i.e., cytoplasmic IgM+, surface immunoglobulin-. Hybridomas were produced against this cell line by fusing spleen cells from hyperimmunized mice with NS-1 mouse myeloma cells. One monoclonal antibody derived from this fusion, designated BA-1, reacted with peripheral blood B lymphocytes, chronic lymphocytic leukemias, pre-B-ALL, most non-Hodgkin's lymphomas, and most non-T, non-B-ALL. BA-1 showed weak reactivity with B lymphoblastoid cell lines and failed to react with multiple myeloma and pokeweed mitogen-induced plasma cells. BA-1 also reacted with peripheral blood granulocytes and the erythroleukemia cell line K-562. No reactivity was seen with cells of T lymphocyte origin, platelets, red cells, monocytes, or acute myelocytic leukemias. Evidence is presented indicating that the determinant recognized by BA-1 is not surface immunoglobulin, HLA-DR, or receptors for C3 or Fc. We conclude that monoclonal antibody BA-1 may be useful in the study of early stages of human B lymphocyte development.

Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein

Abstract: A modified polyoma virus genome has been constructed which can encode the middle-T protein, but not the large-T or small-T proteins. This was achieved, starting with the full length viral DNA inserted into a plasmid vector, by replacing a small genomic restriction fragment spanning the middle-T intervening sequence with the equivalent fragment from a cloned partial cDNA copy of the middle-T protein mRNA. Transfection of the modified viral DNA into cultured rat cells efficiently induced the formation of transformed cell foci which gave rise to cell lines that grew as tumours after injection into Fisher rats. The only viral early-region antigen synthesized by the cell lines was the middle-T protein. Expression of the middle-t protein is therefore sufficient to establish and maintain a transformed state. The viral mRNA produced by two of the transformed cell lines was structurally indistinguishable from the normal middle-T mRNA found in productively infected cells, suggesting that RNA splicing is not an essential step in the biogenesis of this messenger.

Coexistence of neuronal and glial precursor cells in the cerebral ventricular zone of the fetal monkey: an ultrastructural immunoperoxidase analysis

Abstract: The cytological composition of the proliferative zones in the fetal monkey occipital lobe was examined at the light and electron microscopic levels by immunoperoxidase localization of glial fibrillary acid protein (GFA), a protein that is present in astrocytes and radial glial cells but not neurons. During the peak of neurogenesis at embryonic day 80, two distinct classes of proliferative cells, GFA-positive and GFA-negative, are intermixed in the ventricular and subventricular zones. Both cell types are readily recognized in different phases of the mitotic cycle along the ventricular surface. The results indicate that, contrary to prevailing views, (1) glial and neuronal cell lines coexist within the fetal proliferative zones and (2) the onset of glial phenotypic expression occurs prior to the last cell division.

Spontaneous release of a factor with properties of T cell growth factor from a continuous line of primate tumor T cells.

Abstract: A continuous lymphoid cell line had been previously established from a gibbon with spontaneous lymphosarcoma. This cell line, designated as MLA144, when tested after several years in culture was shown to release spontaneously a factor biologically and biochemically similar to human T cell growth factor (TCGF). Conditioned media (CM) from MLA144 cells support growth and DNA synthesis of T cells from humans, several other species of primates, and also from mice and rabbits. The activity in the MLA144 CM is resistant to 60 degrees C and to low and high pH, has a m.w., as determined by gel filtration, of 21,500, elutes from DEAE-cellulose at 0.04 to 0.06 M sodium phosphate buffer, pH 7.6, and has an isoelectric point of about 6.45. Surface-marker analysis of MLA144 cells by rosetting techniques indicates that they are T cells lacking in the receptor for the Fc portion of IgG. The release of TCGF by MLA144 cells should have practical value in terms of ease of TCGF production and should be of great help in the facilitation of studies on the cell biology and molecular biology of TCGF production.

A neoplastic epithelial duct cell line established from an irradiated human salivary gland.

Abstract: Transformed epithelial cells were isolated by using tissue culture techniques from an irradiated human submandibular salivary gland which showed no neoplastic lesion. These cells, carrying colony-forming ability in semisolid agar, formed a semiconfluent monolayer with occasional tubular arrangement. All transformed clones were demonstrated by electron microscopic examination to be only one type of cells having fine structure similar to intercalated duct cells. Of six clones isolated, one clone with stable growth was cultured within the sponge matrix, resulting in formation of duct-like structure with mucinous eosinophilic substance. Moreover, inoculation of the cloned cells into nude mice resulted in a production of adenocarcinoma with solid and trabecular pattern. These findings indicate that a human intercalated duct cell line carrying tumorigenicity is established from a human submandibular salivary gland with an exposure to irradiation.

Human cutaneous T cell lymphoma and leukemia cell lines produce and respond to T cell growth factor

Abstract: Three cell lines of mature T cell origin derived from patients with cutaneous T cell lymphoma-leukemias (CTCL) were found to be constitutive producers of T cell growth factor (L-TCGF). These are the first reported human cell lines which constitutively produce TCGF. Biologically active TCGF could also be eluted from the surface of these cells using an acid glycine buffer under conditions that maintained cell viability, and subcellular fractionation showed that almost all the TCGF activity was associated with the plasma membrane. Over 30 other human hematopoietic cell lines derived from other disorders were unable to produce TCGF even after induction, and their acid eluates did not contain TCGF activity. L-TCGF from CTCL lines had the same biological activity as TCGF obtained from normal leukocytes (N-TCGF) in that they both supported the long-term growth of normal T cells only after the cells were previously activated by antigen or lectin. Both L-TCGF and N-TCGF increased the rate of proliferation of TCGF-independent and TCGF-dependent CTCL cell lines. The same three factor-independent cell lines that released TCGF adsorbed TCGF in a cell-concentration, time-, and temperature-dependent manner. Since the CTCL cell lines produce TCGF, adsorb TCGF, and increase their proliferative rate in response to TCGF or a related molecule, it is suggested that this endogenously produced factor plays a role in maintaining the abnormal proliferation of these cells in culture as permanently growing cell lines independent of exogenous TCGF. However, this does not mean that this is an essential aspect of neoplastic transformation. Since it is unusual to develop these cell lines in the absence of the continuous need for added TCGF, "autostimulation" may be one of the many unusual variant phenotypic properties sometimes associated with neoplastic cells that gives them a selective advantage for in vitro growth.

Anti-transferrin receptor monoclonal antibody and toxin–antibody conjugates affect growth of human tumour cells

Abstract: The development of the monoclonal antibody technique1 has led to renewed interest in whether the growth of tumour cells may be specifically inhibited by antibodies or antibody–toxin conjugates directed against antigenic determinants selectively expressed on tumour cells2–10. We have recently obtained monoclonal antibodies against the transferrin receptor of human cells11,12, which is thought to have an essential role in transport of Fe across the cell membrane and which is selectively expressed on proliferating cells in vivo and in vitro13–17. In some cases, transferrin receptors can be used as a marker to distinguish between tumour cells and normal tissue11,15–17. Here we show that human tumour cells are specifically killed in vitro by anti-transferrin receptor antibody covalently coupled to ricin or diphtheria toxic subunits. In experiments designed to test the effectiveness of these antibody–toxin conjugates in vivo, we found that anti-transferrin receptor antibody alone inhibits the growth of human melanoma cells in nude mice.

Monoclonal antibodies to mammalian neurofilaments

Abstract: Hybrid cell lines secreting monoclonal antibodies against mammalian neurofilaments have been prepared. An improved protocol for the preparation of neurofilaments and methods for the identification and isolation of such hybridomas are presented. The antibodies produced specifically stain only neuronal cell types in both cerebellar sections and culture.

Phenotypic changes of human neuroblastoma cells in culture induced by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate

Abstract: SK-N-SH and SH-SY5Y human neuroblastoma cells treated by 12-)-tetradecanoyl-phorbol-13-acetate(TPA) express morphological and biochemical changes, which indicate that differentiation towards more mature cells has occurred. The most prominent morphological changes were the development in 40-60% of the cells of cell-surface projections longer than 50 micrometers and cytoplasmic neurosecretory granules demonstrated by electron microscopy. At the biochemical level, TPA induced a two-fold increase in the relative activity of neuron-specific enolase and 30- to 40-fold increase in noradrenaline and adrenaline concentrations. A decrease in proliferation rate of TPA-treated cells was observed. The biological effects of TPA were slightly potentiated by nerve growth factor.

Human trophoblast cell-surface antigens defined by monoclonal antibodies.

Abstract: A series of monoclonal antibodies has been raised against the human choriocarcinoma cell-line, BeWo. Four antigens, Trop-1, -2, -3, and -4, are defined on normal and malignant trophoblast cells. Trop-1 and Trop-2 appear to be specifically expressed on syncytio- and cytotrophoblasts, whereas Trop-3 and Trop-4 are also detected on various tumor cell lines, normal lymphocytes, and monocytes. Anti-Trop-1 and anti-Trop-2 antibodies might prove useful for detection and isolation of fetal trophoblast cells circulating in pregnant women's blood and for diagnosis and therapy in patients having choriocarcinomas and other germ-cell neoplasms.

A Multipotential Leukemia Cell Line (K-562) of Human Origin

Abstract: The K-562 leukemia cell line, originally established in our laboratory, has been characterized as an early precursor of the granulocytic series with a block for differentiation. Since K-562 blasts did not differentiate when cultured for 7-8 days in liquid media or 14-16 days in agar-gel an attempt was made to stimulate their potential for spontaneous differentiation by prolonging the time in culture. Inducers of differentiation were not added to the cultures and the cells were studied when they reached the steady state rather than during exponential growth. The cultivation of K-562 cells for 10 to 11 days in media gradually depleted of the essential nutrients needed for cell division induced their differentiation into early precursors of the monocytic, granulocytic, and erythrocytic series. Thus, the peroxidase reaction for hemoglobin demonstrates benzidine-positive material limited to the region of the Golgi apparatus. Analysis of the hemoglobin by isoelectric focusing indicated major bands in th...