Showing papers on "Cell culture published in 1978"

T Cell Growth Factor: Parameters of Production and a Quantitative Microassay for Activity

Abstract: Several soluble factors have recently been associated with the proliferation and differentiation of thymus-derived lymphocytes. One of these factors present in medium conditioned by T cell mitogen-stimulated lymphocytes has the ability to promote the long-term culture of normal and antigen-specific cytotoxic T cells. We report a method to test for this proliferative stimulus in the form of a sensitive microassay based upon the tritiated thymidine incorporation of continuous murine tumorspecific cytotoxic T cell lines (CTLL). The microassay requires microliter quantities of sample fluid and is amenable to quantitative analysis. This highly reproducible, quantitative assay for T cell growth factor (TCGF) has allowed investigation as to the kinetics of TCGF generation and has revealed that T lymphocytes are required for its production. Further investigation has supported the notion that this nonspecies-specific factor is actively removed from tissue culture medium by the proliferation of either T cell mitogen-activated lymphocytes or CTLL.

A better cell line for making hybridomas secreting specific antibodies

Abstract: FUSION of myeloma cells which grow in tissue culture with spleen cells from an immunised mouse provides a general method for obtaining cell lines (hybridomas) which make antibody of the desired specificity1–3. Hybrids derived from these myelomas make the immunoglobulin (Ig) heavy and light chains of the myeloma parent as well as the antigen-specific heavy and light chains of the spleen cell parent. In conditions in which the two heavy and two light chains associate randomly, a hybridoma would make 10 distinct Ig molecules, and the specific antibody would comprise only 1/16 of the total Ig4,5. To obtain hybridomas making only the specific antibodies requires a tumour cell fusion partner that itself makes no Ig but which can nevertheless be fused with spleen cells to obtain hybrids secreting only the specific antibody. We report here the identification of such a cell line, Sp2/0-Ag14.

Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide

Abstract: The tridecamer d(A-A-T-G-G-T-A-A-A-A-T-G-G), which is complementary to 13 nucleotides of the 3'- and 5'-reiterated terminal sequences of Rous sarcoma virus 35S RNA, was added to chick embryo fibroblast tissue cultures infected with Rous sarcoma virus. Inhibition of virus production resulted. The inference emerges that the tridecamer and its counterpart with blocked 3'- and 5'-hydroxyl termini enter the chick fibroblast cells, hybridize with the terminal reiterated sequences at the 3' and 5' ends of the 35S RNA, and interfere with one or more steps involved in viral production and cell transformation. Likely sites of action are (i) the circularization step of the proviral DNA intermediate, and (ii) the initiation of translation, the latter being described in the following communication [Stephenson, M. L. & Zamecnik, P. C. (1978) Proc. Natl. Acad. Sci. USA 75, 285--288].

Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds.

Abstract: A human leukemic cell line (designated HL-60) has recently been established from the peripheral blood leukocytes of a patient with acute promyelocytic leukemia. This cell line displays distinct morphological and histochemical commitment towards myeloid differentiation. The cultured cells are predominantly promyelocytes, but the addition of dimethyl sulfoxide to the culture induces them to differentiate into myelocytes, metamyelocytes, and banded and segmented neutrophils. All 150 clones developed from the HL-60 culture show similar morphological differentiation in the presence of dimethyl sulfoxide. Unlike the morphologically immature promyelocytes, the dimethyl sulfoxide-induced mature cells exhibit functional maturity as exemplified by phagocytic activity. A number of other compounds previously shown to induce erythroid differentiation of mouse erythroleukemia (Friend) cells can induce analogous maturation of the myeloid HL-60 cells. The marked similarity in behavior of HL-60 cells and Friend cells in the presence of these inducing agents suggests that similar molecular mechanisms are involved in the induction of differentiation of these human myeloid and murine erythroid leukemic cells.

Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1).

Abstract: A monoclonal antibody derived by fusion of mouse myeloma cells with spleen cells from a mouse immunized with F9 teratocarcinoma cells is described. This antibody, which reacts with embryonal carcinoma cells of mouse and human origin and with some preimplantation stage mouse embryos, defines an embryonic stage-specific antigen. This stage-specific antigen (SSEA-1) is first detected on blastomeres of 8-cell stage embryos. Trophectodermal cells are transitorily positive; however, each cell in the inner cell mass eventually expresses this antigen.

Isolation of a human prostate carcinoma cell line (DU 145).

Abstract: A long-term tissue culture cell line has been derived from a human prostate adenocarcinoma metastatic to the brain. The cell line, DU 145, has been passaged 90 times in vitro over a period of 2 years. The cells are epithelial, grow in isolated islands on plastic Petri dishes, and form colonies in soft agar suspension culture. Karyotypic analysis demonstrates an aneuploid human karyotype with a modal chromosome number of 64. Distinctive marker chromosomes (a translocation Y chromosome, metacentric minute chromosomes and three large acrocentic chromosomes) have been identified. Electron microscopy of the original tumor tissue and of the tissue culture cell line show a remarkable similarity in cell organelle structure.

Increased superoxide anion production by immunologically activated and chemically elicited macrophages.

Abstract: We studied the capacity of cultured mouse peritoneal macrophages to generate superoxide anion (O(2-)), the initial product of conversion of oxygen to microbicidal species, during phagocytosis of opsonized zymosan or upon contact with the membrane-active agent phorbel myristate acetate (PMA). Macrophages from mice infected with Bacille Calmette-Guerin (BCG) or injected intraperitoneally with thioglycollate broth or endotoxin, released up to 12 times more O(2-) than did resident peritoneal macrophages, depending upon the cell type and whether the stimulus was zymosan or PMA. There was little if any O(2-) release from resting (unstimulated) macrophages. The density of cells on culture dishes was an important variable since crowding of the dish markedly reduced the efficiency of O(2-) production. The enhanced O(2-) release of chemically elicited and infection-activated macrophages was noted after stimulation with a wide range of concentrations of PMA and zymosan, at all time points studied (up to 120 min), and with cells maintained for 140 rain to 16 days in culture. The O(2-) response of resident cells improved twofold to zymosan and ninefold to PMA during the first 3 days in culture. The capacity to release O~ appears to be limited to actively phagocytic cell types: murine macrophage-like tumor lines and cultured human monocytes released O(2-) when stimulated by PMA or zymosan, fibroblast and endothelial lines and embryo-derived cells did not. Activity of superoxide dismutase, which removes O(2-), was not detectable in culture supernates of any cell type, and thus, differences in detectable O(2-) could not be attributed to variations in the release of this enzyme. We conclude that the phagocytosis- associated respiratory burst is significantly enhanced in mononuclear phagocytes obtained ai~r chemical inflammation or BCG infection. Increased capacity to generate O(2-) and other oxygen radicals during phagocytosis could contribute to the improved microbicidal and tumoricidal activity of activated macrophages.

Anti-viral activity induced by culturing lymphocytes with tumor-derived or virus-transformed cells. Enhancement of human natural killer cell activity by interferon and antagonistic inhibition of susceptibility of target cells to lysis.

Abstract: Interferon, induced in lymphocytes either with viruses or cell lines, increases severalfold the natural cytotoxicity of human lymphocytes on target cell lines. Cell separation experiments support the hypothesis that interferon enhances the activity of natural killer cells rather than generating a new population of effector cells. In mixed culture of lymphocytes and cell lines in which endogenous interferon is produced, interferon mediates an enhancement of cytotoxicity that represents up to 70-90% of the observed cytotoxicity. The effect of interferon on target cells is antagonistic to the effect on the lymphocytes: the susceptibility to cell-mediated lysis of various cells upon pretreatment with interferon is decreased and in some cases almost completely suppressed. Interferon renders target cells resistant to natural killer cells acting by an intracellular mechanism which requires RNA and protein synthesis. While normal fibroblasts are protected, virus-infected cells and most tumor cells usually are not protected by interferon. Interferon by stimulating very efficient nonspecific cytotoxic cells and by protecting at the same time normal cells from lysis, might render the natural killer cell system an inducible selective defense mechanism against tumor and virus-infected cells.

Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides.

Abstract: The chemotactice response of human dermal fibroblasts of type I, II, and III human collagens and collagen-derived peptides was quantitated by an in vitro assay. All three native human collagens and constituent alpha chains can serve as chemoattractants for fibroblasts in vitro. When type I, II, and III collagens were digested by bacterial collagenase, the resulting peptides were also chemotactic. In addition, synthetic di- and tripeptides containing hydroxyproline were also chemotactic for fibroblasts. Since collagen is degraded and remodeled at sites of tissue injury and inflammation, these findings suggest that collagen and collagen-degradation peptides might function as chemotactic stimuli for fibroblasts in vivo and attract these cells to effect repair of damaged tissue.

Heterogeneity of tumor cells from a single mouse mammary tumor.

Abstract: By the use of a variety of cell culture and separation methods, four cell lines were isolated from a single autochthonous BALB/cfC3H mammary tumor. These lines differ markedly from each other in culture morphology, various in vitro growth properties, expression of murine mammary tumor virus antigen, and karyotype, yet all four lines are tumorigenic in normal, syngeneic hosts, yielding tumors of generally similar histology, although distinct from the original neoplasm. Three of the four lines have been cloned from soft agar. The clones exhibit the same growth properties as the lines from which they were derived. Karyotypic analysis of the parent tumor revealed the presence of cells with heterogeneous numbers of chromosomes similar to those seen in the isolated lines, suggesting both the presence of these distinct cell types in the original neoplasm and a genetic origin of the diversity.

Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431.

Abstract: The binding and internalization of epidermal growth factor (EGF) in human epithelioid carcinoma cells (A-431), which have approximately 2.6 X 10(6) receptors per cell, has been followed with 125I-labeled EGF and by fluorescence microscopy. We have prepared a fluorescent derivative of EGF that is biologically active and retains substantial binding affinity for cell receptors. After binding of this derivative to cells at 6 degrees, the cellular borders were prominently stained and the fluorescence on the remainder of the membrane was uniform. Upon warming of these cells to 37 degrees for 10 min, the surface fluorescence diminished and randomly distributed endocytotic vesicles appeared in the cytoplasm. After 20 min at 37 degrees these fluorescent vesicles formed a perinuclear ring. The binding of EGF to the surface of these cells was also visualized by immunofluorescence using rabbit antibodies to EGF and rhodamine-labeled goat anti-rabbit antibodies. We did not detect large fluorescent clusters or cap formation in these experiments. These data provide direct confirmation of the previous biochemical data that suggested that cell membrane-bound EGF is rapidly internalized.

Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity

Abstract: A permanent human cell line that maintains the granulocytic characteristics of acute myelogenous leukemia cells has been established. The cells of this line form myeloid colonies in soft gel culture in the presence of human colony-stimulating activity. The cell line may be useful for studying human acute myelogenous leukemia and the mechanism of response to colony-stimulating activity.

Method of producing antibodies

Abstract: Continuous cell lines of genetically stable fused cell hybrids capable of producing large amounts of monoclonal antibodies against specific viruses and their antigenic determinants have been developed. The cell lines are fused cell hybrids between viral antibody producing cells and myeloma cells. Fused cell hybrids between influenza virus-primed mouse spleen cells and mouse myeloma cells can be maintained indefinitely in culture and continue to produce large amounts of anti-influenza antibody.

Nerve growth factor prevents the death and stimulates the neuronal differentiation of clonal PC12 pheochromocytoma cells in serum-free medium

Abstract: The PC12 clone is a noradrenergic cell line derived from a rat pheochromocytoma. In culture medium containing horse serum, PC12 cells undergo mitosis; when nerve growth factor (NGF) is included in the medium, the cells cease multiplication and extend neuritis. It is shown here: (a) that PC12 cells are not viable in serum-free medium. When serum is withdrawn, 90 percent of the cells die within 4-6 days and 99 percent by 2-3 wk. (b) If NGF is added at the time of serum withdrawal, the cells undergo one doubling and remain viable for at least 1 mo. (c) Addition of NGF to cultures after more than 2 days in serum-free conditions results in maintenance of surviving cells, but not in an increase in cell number. (d) NGD also induces neurite outgrowth from PC12 cells in serum-free medium. (e) NGF-treated cells exhibit much less cell-cell and neurite-neurite aggregation in the absence than in the presence of serum. (f) The apparent minimum level of 2.5S NGF required for PC12 survival and morphological differentiation in serum-free medium is about 10 ng/ml (approximately 0.4 nM). (g) Withdrawal of NGF in serum-free conditions results in degeneration of neurites and loss of cell viability. (h) Experiments with campotothecin demonstrate that the effects of NGF on survival and neurite outgrowth may be uncoupled and suggest that the survival effects are transcriptionally independent. The present results also suggest that PC12 cells have a requirement for NGF (similar to that of normal sympathetic neurons) and that serum may substitute for this requirement. In addition, the present system of maintaining a highly differentiated cell line in a chemically defined medium suggests certain experimental opportunities.

Selection and characterization of bovine aortic endothelial cells

Abstract: This paper reports techniques for isolation, selection and long-term passage of bovine aortic endothelium (BAE). A [3H]thymidine-selection technique was developed to limit overgrowth of cultures by contaminating smooth-muscle cells. The resulting cultures could be passaged for a replicative life span of 35 to 40 doublings and maintained a stable, normal karyotpye throughout this period. Despite the fact that these cultures reached a stable monolayer with density-inhibited growth state, postconfluent cells showed focal areas of a second growth pattern called "sprouting." This was seen only when cultures were maintained at high densities for periods of 1 to 2 weeks. Ultrastructural analysis, as well as immunofluorescence studies with markers for endothelial cells (factor VIII) and smooth-muscle cells (actin), indicates that this phenomenon is not due to overgrowth of a residual population of smooth-muscle cells, but may represent a second growth pattern of the endothelial cells themselves.

Human breast carcinoma cells in continuous culture: a review.

Abstract: A comprehensive listing of putative human breast carcinoma cell lines and the extent to which each has been characterized is presented. Criteria used to certify the human, mammary, and malignant origin of a cell line include: ( a ) a reliable histopathological diagnosis; ( b ) interspecies specificity established by human karyotype, isoenzyme profiles, and/or cell surface antigenicity; ( c ) intraspecies specificity, demonstrated by genetic evidence of a unique, human donor distinct from other cells including HeLa cells; and ( d ) organ specificity, supported by morphological evidence of epithelial structure and secretory activity, and especially by the expression of differentiated functions; these include presence of receptors for sex steroid hormones, hormone responsiveness, and production of milk proteins, fatty acids, or milk-specific antigens. Of the 47 cell lines for which data are here reported, 22 have been shown to be derived from human, non-HeLa donors and to have epithelial morphology as revealed by light or electron microscopy. Differentiated function has been recorded for 19 cell lines. Additional human breast cancer cell lines have been reported, but characterization of some of these has been insufficient to judge the legitimacy of their pedigrees. For others mammary origin is questionable. Six purported breast cell lines are in reality HeLa cells, and one is of nonhuman origin.

Murine B Cell Leukemia Line with Inducible Surface Immunoglobulin Expression

Abstract: A chemically induced leukemia of BDF 1 mice, designated 70Z/3, was adapted to tissue culture in our laboratory A minority of these cells displayed surface immunoglobulin (sIg) as detected by immunofluorescence with rhodamine-labeled class (IgM)-specific antibodies Addition of the B cell mitogen, LPS, to the cultures induced sIg expression on all of these cells The kinetics of this transition were dependent on the dose of LPS As little as 01 µg/ml induced sIg on >97% of the cells within 36 hr DxS also induced sIg whereas other mitogens (Con A, PPD) or inducing agents (DMSO, dimethyl formamide, butyric acid), tested over a wide range of concentrations, failed to induce sIg expression The cells bear H-2 antigens but lack IgD, IgG, IgA, Ia, and Thy 12 Exposure to LPS had no effect on the presence or absence of these structures A small percentage of cells possessed receptors for complement and for the Fc portion of immunoglobulin Cytoplasmic IgM was detectable within all of the cells, and constitutive production of small quantities of IgM was confirmed by SDS-polyacrylamide gel electrophoresis of serologic precipitates of cell lysates after pulsing with radioactive amino acids Using similar methods, we failed to detect active secretion of immunoglobulin Thus, this cell line has properties similar to cytoplasmic Ig + , surface Ig - cells found in immature tissues and in bone marrow of mice and humans that are thought to be immediate precursors of sIg + B lymphocytes It may provide a model for studying the mechanism of LPS activation and the molecular events associated with externalization of cell surface receptors

Cell lineages of the embryo of the nematode Caenorhabditis elegans.

Abstract: Embryogenesis of the free-living soil nematode Caenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock--i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. Embryogenesis was followed microscopically with Nomarski interference optics and high-resolution video recording.

Anti-viral activity induced by culturing lymphocytes with tumor-derived or virus-transformed cells. Identification of the anti-viral activity as interferon and characterization of the human effector lymphocyte subpopulation.

Abstract: A viral inhibitor(s) is released in the supernate of mixed cultures containing human or mouse lymphocytes and cells from certain lines. The inhibitor is active against a variety of unrelated viruses and is a protein that is not toxic for cells. It does not inactivate viruses directly, but inhibits viral replication through an intracellular mechanism that involves synthesis by the cells of both RNA and protein. These characteristics identify the inhibitor as an interferon. The anti-viral activity is contained in at least two molecular species, of approximately 25,000 and 45,000 daltons, respectively. In addition to the anti-viral activity, the supernates of the mixed cultures display an anti-cellular activity, the inhibition of DNA synthesis and of cell multiplication. The anti-viral and the anti-cellular activities are positively correlated in supernates from various cultures and in partially purified preparations. The human cell population responsible for interferon production is composed mainly of Fc-receptor positive, surface immunoglobulin negative, non-T-cell lymphocytes. The ability of certain cell lines to induce interferon seems to be preferentially associated with tumor origin or with in vitro transformation by certain viruses (Epstein-Barr virus, murine sarcoma virus).