Showing papers on "Cellular differentiation published in 1981"

Differentiation of mouse myeloid leukemia cells induced by 1 alpha,25-dihydroxyvitamin D3

Abstract: Mouse myeloid leukemia cells can be induced to differentiate into macrophages in vitro by 1 alpha,25-dihydroxyvitamin D3, the active form of vitamin D3. The minimal concentration of 1 alpha,25-dihydroxyvitamin D3 to induce the cell differentiation was 0.12 nM. The degree of cell differentiation in various markers induced by 12 nM 1 alpha,25-dihydroxyvitamin D3 was nearly equivalent to that induced by 1 microM dexamethasone, the most potent known stimulator. Among several markers of the differentiation by 1 alpha,25-dihydroxyvitamin D3, phagocytic activity was induced within 24 hr, and this was followed by induction of lysozyme and locomotive activities. Similar changes were also induced by 0.01-1 microM 1 alpha-hydroxyvitamin D3. 25-Hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 showed only weak inducing activity. These results suggest the possibility that, in addition to its wellknown biological activities in enhancing intestinal calcium transport and bone mineral mobilization, 1 alpha, 25-dihydroxyvitamin D3 is involved in the differentiation of bone marrow cells.

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.

Isolation and characterization of human muscle cells.

Abstract: We have developed an in vitro system for the study of postnatal human muscle under standardized conditions. The technique utilizes cloning to isolate pure populations of muscle cells. By manipulating culture conditions we can maximize either proliferation or differentiation of individual clones or of clones pooled to yield mass cultures of muscle cells. The muscle phenotype is stable; cells can be stored in liquid nitrogen for long-term use without loss of proliferative or differentiative potential. We have determined proliferative capacity of muscle cells from an analysis of clonal growth kinetics; we determined differentiative capacity from morphological evidence (cell fusion, striations, contractions, and the appearance of acetylcholine receptors) and biochemical analysis of muscle protein synthesis (creatine kinase, alpha-actin, tropomyosin, and myosin light chains). Our approach eliminates the variability in cellular composition that has complicated studies of primary muscle to date. We can now study in a controlled fashion the interactions and contributions of different cell types to the development of normal and genetically dystrophic human muscle.

Cell interactions modulate embryonal carcinoma cell differentiation into parietal or visceral endoderm

Abstract: F9 is a clonal line of mouse teratocarcinoma-derived embryonal carcinoma (EC) cells which shows very little spontaneous differentiation in vivo or in vitro1. Recently, however, it was reported2–6 that F9 monolayers treated with retinoic acid and dibutyryl cyclic AMP differentiate into an early embryonic cell type known as parietal endoderm, which is one of two distinct populations of extra-embryonic endoderm that differentiate in the normal mouse embryo shortly after implantation7,8. The other population is the visceral endoderm, and the two differ not only in their morphology and location within the embryo, but also in their biochemical properties9–12. Parietal endoderm cells, for example, do not synthesize α-fetoprotein13 (AFP), whereas visceral endoderm cells do13. There is evidence8 that during embryogenesis parietal and visceral endoderm are derived from a common precursor population known as the primary endoderm, and recent experiments with cultured mouse embryos14 suggest that the phenotype of these cells can be modulated by contact with different embryonic tissues. We now show that if F9 EC cultures are treated with retinoic acid when they are in the form of small aggregates, they differentiate on the outer surface cells which morphologically resemble visceral rather than parietal endoderm. In addition, the cells synthesize and secrete AFP, identified by immunoprecipitation and immunoperoxidase reactions using specific anti-AFP immunoglobulin. One interpretation of this result is that F9 cells treated with retinoic acid differentiate first into multipotent cells analogous to the primary endoderm of the normal embryo which then express either the mature parietal or visceral phenotype depending on the nature of the intercellular contact signals they receive. We therefore believe that F9 EC cells may be even more useful than previously supposed for biochemical studies on factors controlling gene expression during mammalian embryogenesis.

Inducer T lymphocytes synthesize a factor that stimulates proliferation of cloned mast cells

Abstract: Inducer T lymphocytes activate other cells to divide and express new function. Known target cells include other lymphocytes and haematopoietic stem cells1. We now provide evidence that the inducer T cell acts on another important target population: mast cells. Mast cells have a central role in the expression of immediate hypersensitivity and are also prominent in T-cell mediated reactions of the delayed type2–7. Because the proliferation of differentiated cells is often regulated by soluble growth factors, we examined an inducer T-cell clone for its ability to stimulate mast cell proliferation. We report here that cloned Ly1+2− inducer T cells produce a factor that selectively induces morphologically and karyotypically normal mouse mast cell clones to proliferate. We therefore suggest that inducer T cells may regulate mast cell numbers by releasing a soluble growth factor that stimulates them to divide. Because mast cell products also affect certain T-cell functions8–10, mast cell-T cell interactions may comprise part of an immunoregulatory circuit.

Cell Replacement in Epidermis (Keratopoiesis) via Discrete Units of Proliferation

Abstract: Publisher Summary This chapter reviews cell replacement in epidermis (keratopoiesis) via discrete units of proliferation. Dorsal or ear epidermis from mice is studied. For mouse epidermis, the data indicate that the tissue is subdivided into discrete units of proliferation, each with a certain autonomy but not independent of the neighboring units because they have to interact to produce an effective surface barrier. Each unit is maintained and dependent upon a group of about ten basal cells, one of which would appear to be a stem cell. The size and topographical relationship between the cells is illustrated in the chapter. The chapter also discusses a two-tier proliferative system; with this system, controls may operate at many levels. Any of the controls could potentially be changed; the chapter discusses several related consequences. The rate of progression of the stem cell through the cycle could be altered because of changes in its position or as a consequence of changes in the levels of, or susceptibility to, those factors that may act as on/off switches. Several possible changes might occur in the transit population, which can have dramatic effects on the total cell output.

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...

Differential and synergistic actions of nerve growth factor and cyclic AMP in PC12 cells.

Abstract: When a clonal line of rat pheochromocytoma (PC12) was exposed to beta-nerve growth factor (beta NGF), N6, O2-dibutyryl adenosine 3':5' cyclic monophosphate (Bt2cAMP), or a combination of the two, 10, 26, or 70% of the cell clumps, respectively, displayed neurites after 1.d. Increases in the cellular RNA concentration were also found to be additive or greater when both agents were present. Neurites induced by Bt2cAMP alone were not maintained after replacement with beta NGF. The degree of potentiated neurite outgrowth was a function of the time of simultaneous exposure to both agents. The initiation of neurite outgrowth in the presence of Bt2cAMP was independent of RNA synthesis, in contrast to that induced by beta NGF alone. We conclude that beta NGF-induced initiation of morphological differentiation of these cells is not mediated by a cAMP-dependent mechanism. Consideration of Bt2cAMP effects upon other cell lines suggest that Bt2cAMP causes a rapid, but unstable, reorganization of the PC12 cytoskeleton, resulting in the initiation of neurite outgrowth from these cells. In contrast, beta NGF alone achieves a more stable cytoskeleton reorganization by an RNA synthesis-dependent mechanism.

Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor

Abstract: PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.

Sex hormone regulation of in vitro immune response. Estradiol enhances human B cell maturation via inhibition of suppressor T cells in pokeweed mitogen-stimulated cultures.

Abstract: The effects of the main male and female sex hormones, testosterones and estradiol, in pokeweed mitogen (PWM)-stimulated cultures of human blood lymphocytes were studied. We found that the addition of physiological concentrations of estradiol (780-2,600 pmol/liter) to PWM cultures significantly increased the accumulation of immunoglobulin M-containing and -secreting cells detected by immunofluorescence and/or by the reversed protein-A plaque assay. The dose range of estradiol that induced enhanced B cell maturation did not affect the proliferative response. Estradiol displayed the same effect in vitro on lymphocytes from both men and women. Fractionation of lymphocyte subpopulations before culturing revealed that estradiol does not display a direct mitogenic or stimulatory effect of B cells. Instead, estradiol inhibits the suppressive activity of a radio-sensitive (1,000 rad) subset of T lymphocytes bearing Fc-receptors for immunoglobulin G. Nontoxic concentrations fo testosterone did not influence the in vitro B cell maturation. These observations provide a cellular basis for the differences in the immunoreactivities of males and females. The estradiol-induced inhibiton of suppressor T cells might be important for the pathogenesis of various autoimmune disorders.

A monoclonal antibody that recognizes B cells and B cell precursors in mice.

Abstract: The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

Expression of the macrophage-specific antigen F4/80 during differentiation of mouse bone marrow cells in culture.

Abstract: We have defined the expression of the macrophages (m phi)-specific antigen (Ag) F4/80 during differentiation in culture. The progenitor cells-the colony-forming unit in culture and cluster-forming cell-lacked Ag F4/80 but gave rise to colonies of F4/80-positive adherent m phi, as shown by fluorescence-activated cell sorting and clonal assays with L cell-conditioned medium as the source of growth factor. Ag F4/80 first appeared on a nonadherent precursor found in mass liquid BM cultures after 3 d. Once adherent, m phi expressed high levels of Ag F4/80 and other markers. The role of L cell-conditioned medium and of adherence on expression of Ag F4/80 was also examined. Clonal analysis of F4/80 and other Ag, Mac-1, and 2.4G2 (FcR) showed that all cells in all independent colonies come to express these markers. These studies establish that F4/80 is a marker for the more mature stages of m phi development and that Ag expression increases progressively during maturation in vitro. Heterogeneity of Ag expression can be ascribed to variation in development and not to independent subsets of the m phi.

Mouse skin cells resistant to terminal differentiation associated with initiation of carcinogenesis

Abstract: In chemical carcinogenesis, initiation is defined as the exposure of a tissue, generally mouse skin, to a limited dose of carcinogen which by itself does not induce tumours1. Subsequent treatments, usually by tumour promoters, are required to allow expression of initiation. In the absence of further treatment, the initiated cells are not recognizable by any known biochemical or biological criteria and the tissue itself functions normally. Much research has centred on elucidating the mechanism of initiation1–3. It seems likely that initiation involves a genetic change in the target cell as its expression after tumour promotion is a permanent characteristic of the tissue, even if many cell generations have elapsed between initiation and promotion4. Studies on tissues from patients with familial polyposis coli suggest that the occurrence of initiated cells in these individuals is due to an autosomal dominant gene5. Few studies have actually attempted to define the biological nature of the initiating alteration itself. We report here that cells resistant to terminal differentiation can be readily isolated from skin of BALB/c mice exposed to an initiating dose of carcinogen in vivo but not from control mouse skin. Also, we found that one strain of mouse (SENCAR) might contain a preexisting population of cells resistant to differentiation, and possibly initiated for tumorigenesis.

Stages of B cell differentiation in human lymphoid tissue.

Abstract: Monoclonal antibodies reactive with B cell-specific differentiation and other antigens were used to investigate stages of B cell maturation in human lymphoid tissue, using an immunoperoxidase technique on frozen tissue sections. Lymphoid follicles, which represent the major anatomic compartment of B cells, demonstrated cellular antigenic expressions that appear to reflect differentiation of B cells. The majority of cells in the primary follicles and the mantle zones of secondary follicles expressed surface antigens similar to those of circulating B cells, namely IgM, IgD, Ia, B1, and B2. In contrast, the germinal center cells of secondary follicles stained for IgM, IgG, B1, B2, and Ia antigens, but not for IgD, and furthermore, acquired the T10 antigen. The germinal centers stained much more intensely than mantle zones with anti-B2, whereas no such striking difference in the staining intensity was observed with anti B1. Plasma cells, which represent the end stage of B cell differentiation, showed intense cytoplasmic staining with the anti-T10 antibody. The results indicate that the generation of germinal center cells in primary lymphoid follicles involves phenotype changes that correspond largely to those previously observed after both antigenic and mitogenic activation of B lymphocytes.

The persisting (p) cell: histamine content, regulation by a t cell-derived factor, origin from a bone marrow precursor, and relationship to mast cells.

Abstract: Histamine was detected at levels of 100 ng/10(6) cells in the metachromatic granules of the persisting (P) cell, which appears in cultures of murine lymphoid or bone marrow cells and is capable of long-term growth in vitro in the presence of a T cell-derived growth factor. This factor, which we termed P-cell stimulating factor, was distinct from t-cell growth factor and had an apparent molecular weight of 25,000-30,000. P cells did not originate from Thy.1-positive cells nor was the thymus necessary for the development of their precursors. Moreover, P cells grew directly from colonies generated in agar cultures of bone marrow cells, the nature of the colonies indicating that P cells shared a common precursor with hemopoietic cells. Mutant Wf/Wf mice, although deficient in certain mast cells, possessed P-cell precursors. It is hypothesized that P cells are related to a specialized subset of mast cells, derived from a bone marrow progenitor but regulated by activated T cells.

Functional analysis of human T cell subsets defined by monoclonal antibodies. IV. Induction of suppressor cells within the OKT4+ population.

Abstract: In this report, we explored the functional heterogeneity within the OKT4+ subset of human T cells. Evidence was obtained that although in vitro pokeweed mitogen-activated OKT4+ cells can function as radioresistant helper cells, these activated OKT4+ cells could also exert potent feedback suppression. Despite the induction of suppressor cells after pokeweed mitogen activation, the OKT4+ population maintains its original OKT3+, OKT4+, nd OKT8- surface phenotype. The suppressor cells contained within the activated OKT4+ population were found to be radiosensitive. Importantly, the suppression mediated by activated OKT4+ cells required the presence of radiosensitive cells contained within the resting OKT4+ population. Taken together, these results suggest that the OKT4+ subset of human T cells contains cells that can be activated to differentiate into suppressor cells independent of OKT8+ cells.

Lonidamine, a Selective Inhibitor of Aerobic Glycolysis of Murine Tumor Cells

Abstract: The action of Lonidamine [1-(2,4-chlorobenzyl)-1H-indazol-3-carboxylic acid] on respiration and aerobic lactate production of several murine tumor cells and normal differentiated murine cells was investigated. Lonidamine reduced the oxygen consumption in both normal and neoplastic cells. In contrast, it increased the aerobic glycolysis of normal cells but inhibited that of tumor cells. This selective action might be ascribed to the inhibition of mitochondrially bound hexokinase, which is usually absent in normal differentiated cells.

The stem‐cell zone of the small intestinal epithelium. III. Evidence from columnar, enteroendocrine, and mucous cells in the adult mouse

Abstract: In the first two articles of this series we demonstrated restriction of Paneth cell formation to positions 5 and above. Restriction was independent of the Paneth cell population-density gradient in the crypt base. We concluded that our results were consistent with the presence in the adult of a stem-cell zone in positions 1–4 in which stem cells received no inducement to differentiate. To further test the stem-cell zone hypothesis we determined the site of stem-cell differentiation along mucous, enteroendocrine, and columnar cell lines using radioautography with 3H-thymidine, as a label. One hour after injection of 3H-thymidine, labeled mucous cells were not observed below position 5. Only later did they appear in lower positions and not until 4 days after injection were they observed in position 1. Labeled enteroendocrine cells first appeared above, and then were seen in the top of, and finally in the middle and bottom of the Paneth cell distribution. Thirty hours after injection there were two populations of labeled columnar cells in the crypt base, a heavily labeled population and a lightly labeled one. At this time interval the heavily labeled columnar cells were only observed in position 5 and above, but they appeared in position 1–4 by 66 hours after injection. The above evidence led us to conclude that all differentiated offspring of the common epithelial stem cell originate in positions 5 and above. Most columnar, mucous, and enteroendocrine cells originating in positions 5 and above migrate upward. However, some of these cells migrate down. All differentiated cells found in positions 1–4 migrated down from their origin in position 5 or above. We also found that only stem cells proliferate in positions 1–4. We concluded that in the adult, there is a stem-cell zone in positions 1–4 where stem cells are not induced to differentiate and persist as stem cells throughout life.

Long-term in vitro culture of murine mast cells. I. Description of a growth factor-dependent culture technique.

Abstract: A feeder layer independent long-term in vitro culture system for murine mast cells is described. Concanavalin A-activated murine splenic leukocyte-conditioned medium, prepared under conditions optimal for T cell growth factor production, has been found also to contain a growth-promotion activity for murine mast cells identified by their morphology, characteristic ultrastructure of the granules, positive reactions with toluidine blue and alcian blue, presence of receptors for IgG and IgE, as well as presence of histamine, serotonin, L-Dopa, 5-hydroxytryptophan, and sulfated products within the cytoplasm. After 2 to 3 wk of culture in the presence of the conditioned medium, mast cell lines were established from various sources initially devoid of matured mast cells. Such sources included spleen and bone marrow of athymic nude mice, long-term cultured marrow cells as well as T cell-depleted normal marrow. Cultured mast cells are absolutely dependent upon the conditioned medium-derived growth factor(s) for growth and viability. Death ensues within 24 hr in the absence of the factor(s). Established mast cell lines have been maintained in exponential growth for over 1 yr by passaging in the conditioned medium every 3 to 7 days.

Human Squamous Cell Carcinoma: Establishment and Characterization of New Permanent Cell Lines

Abstract: • Squamous cell carcinoma is the most common of human cancers, and yet because it is poorly represented by cultured cell lines, little is known about the characteristic cell biology and the cellsurface antigenic phenotypes of such tumors. To develop a continuously available source of squamous cell carcinoma for repeated and reproducible serologic analysis and for better understanding of its biologic characteristics, tissue culture methods and nude mice were used to establish new cell lines of squamous carcinoma. Special media, serum supplements from several sources, and methods of handling fresh tissue specimens were all examined as a means of improving the survival of tumor cell lines. Several new cell lines were established. Features characteristic of a squamous cell origin, eg, microvilli, desmosomes, tonofilaments, and the squamous cell differentiation antigen (pemphigus antigen), were found. The clinical course of disease in individual donor patients has been examined. ( Arch Otolaryngol 1981;107:703-710)

Embryonic erythroid differentiation in the human leukemic cell line K562

Abstract: K562 human leukemia cells synthesize embryonic hemoglobins after culture in the presence of hemin. We have rigorously identified these hemoglobins by globin chain analysis and peptide mapping. No adult hemoglobin could be detected, and beta-globin synthesis was less than 2 ppm of total protein synthesis. Persistent embryonic globin gene expression is known to occur as a consequence of globin gene deletions. However, restriction endonuclease mapping showed that the globin gene complexes in K562 cells are indistinguishable from normal. Hemin increased the rate of embryonic globin synthesis. The pattern of hemoglobin synthesis proved to be stable when cells from different laboratories were compared. One line, however, synthesized large amounts of Hb X and very little Hb Portland in response to hemin. Hb X has been previously detected in human embryos; we show here that it has the composition epsilon 2 gamma 2 and is diagnostic of imbalanced chain synthesis or "zeta thalassemia." We have identified several agents that induce hemoglobin synthesis in K562 cells. Different inducers induced different patterns of embryonic hemoglobin synthesis but never any adult hemoglobin synthesis.