Showing papers on "Cell growth published in 1980"

Inhibition of rat arterial smooth muscle cell proliferation by heparin. In vivo studies with anticoagulant and nonanticoagulant heparin.

Abstract: Heparin inhibits the proliferation of intimal smooth muscle cells which occurs after denudation of endothelium by air-drying injury in the rat carotid artery. We determined (1) whether the antiproliferative effect of heparin is secondary to effects on platelet adherence to subendothelium or endothelial regeneration and (2) whether the antiproliferative and anticoagulant activities of heparin are related. Morphometric observations by scanning electron microscopy showed that heparin did not alter platelet adherence 5 days after arterial injury and had little or no effect on endothelial regener- ation at 5 and 10 days. To study the relationship between the antiproliferative and anticoagulant effects, we fractionated heparin by affinity chromatography on antithrombin-Sepharose into purified anticoagulant and nonanticoagulant fractions. These heparin fractions were administered to rats in doses which were equivalent either in terms of anticoagulant activity or in terms of mass to the dosage of unfractionated heparin known to inhibit myointimal growth. Additionally, some rats received nonanticoagulant heparin at a dose which was greater in terms of mass than the highest dose of unfractionated heparin which could be administered without inducing fatal hemorrhage. Inhibition of myointimal growth, determined by morphometric analysis of total plaque volume 2 weeks after arterial injury, correlated with total mass of heparin administered but not with anticoagulant activity. Non- anticoagulant heparin given at high dose caused 77% inhibition of myointimal growth (P = 0.02 vs. controls). Heparin inhibition of arterial smooth muscle cell proliferation does not appear to be mediated either by effects on other cells at the level of the arterial wall or by antithrombin. This study should direct attention toward a potential growth regulatory role for arterial glycosaminoglycans. Circ Res 46: 625-634, 1980

Cell proliferation and migration on collagen substrata in vitro

Abstract: Quantitative data are presented regarding cell proliferation and migration on (a) collagen films (b) the surface of 3-dimensional gels of native collagen fibres and (c) within the 3-dimensional collagen gel matrix, as part of a study of the effects of the extracellular matrix on cell behaviour. The nature of the collagen environment was found to influence the proliferation of certain cell types, but not of others. For example, HeLa cells proliferate at approximately the same rate and reach the same saturation cell densities on all of the collagen substrata, while human skin fibroblasts grow more slowly within the 3-dimensional collagen gel matrix compared with cells either on the gel surface or on collagen films. The 3-dimensional gels of native collagen fibres may also be used to study cell migration on the gel surface, as well as cell migration (or 'infiltration') from the gel surface into the 3-dimensional collagen matrix. Two methods have been used to obtain quantitative information concerning cell infiltration into the collagen gel, one involving the selective removal of cells from the gel surface, while the other relies on direct microscopic examination. Of the cells examined to date, epithelial cells (both normal and tumour) do not show infiltrative behaviour, while both normal and virally transformed fibroblasts, as well as tumour cells of non-epithelial origin (e.g. melanoma), do infiltrate into the collagen gel matrix, at rates which vary considerably according to cell type.

Inhibition of rat arterial smooth muscle cell proliferation by heparin. II. In vitro studies.

Abstract: We studied in vitro the effects heparin on the growth of rat aortic smooth muscle cells. Measurements of growth were monitored by [3H]thymidine uptake and changes in cell number over a period of 3 days. Our results show that heparin-highly anticoagulant or nonanticoagulant-significantly inhibits growth of smooth muscle cells. We also show that this is a highly specfic interaction with regard to molecule and cell type: i.e., other polyanions, except for a low molecular weight dextran sulfate, do not have the same effect on growth, and not all cells are inhibited by heparin; e.g., endothelial cell growth actually is enhanced. After removing antithrombin from our media, we carried out experiments which show that heparin is effective even though thrombin, a potent mitogenic agent, is still present and active. We also found that passing the platelet extract over a heparin column did not remove all of the motogenic activity of the platelet preparation. Both experiments indicate an inhibitory role for the heparin molecule, per se. Our results support the findings of a recent paper (Guyton et al., 1980) showing that heparin can limit the size of myointimal plaques in rats after carotid injuries by inhibiting smooth muscle cell proliferation.

Evaluation of cytotoxicity in cultured cells by enzyme leakage

Abstract: The measurement of cell injury or cytotoxicity produced by chemicals or toxicants in cell culture has routinely been evaluated by such criteria as plating efficiency, cell growth, protein or DNA synthesis, and cell viability. We describe a more sensitive and earlier index of cytotoxicity: leakage of cytoplasmic enzymes from injured cells into the culture medium.

Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhibition of cell proliferation

Abstract: GH(4)C(1) cells are a clonal strain of rat pituitary cells that synthesize and secrete prolactin and growth hormone. Chronic treatment (longer than 24 h) of GH(4)C(1) cells with epidermal growth factor (EGF) (10(-8) M) decreased by 30-40 percent both the rate of cell proliferation and the plateau density reached by cultures. Inhibition of cell proliferation was accompanied by a change in cellular morphology from a spherical appearance to an elongated flattened shape and by a 40-60 percent increase in cell volume. These actions of EGF were qualitatively similar to those of the hypothalamic tripeptide thyrotropin-releasing hormone (TRH) (10(-7) M) which decreased the rate of cell proliferation by 10-20 percent and caused a 15 percent increase in cell volume. The presence of supramaximal concentrations of both EGF (10(-8)M) and TRH (10(-7)M) resulted in greater effects on cell volume and cell multiplication than either peptide alone. EGF also altered hormone production by GH(4)C(1) cells in the same manner as TRH. Treatment of cultures with 10(-8) M EGF for 2-6 d increased prolactin synthesis five- to ninefold compared to a two- to threefold stimulation by 10(-7) M TRH. Growth hormone production by the same cultures was inhibited 40 percent by EGF and 15 percent by TRH. The half- maximal effect of EGF to increase prolactin synthesis, decrease growth hormone production, and inhibit cell proliferation occurred at a concentration of 5 x 10 (-11) M. Insulin and multiplication stimulating activity, two other growth factors tested, did not alter cell proliferation, cell morphology, or hormone production by GH(4)C(1) cells, indicating the specificity of the EGF effect. Fibroblast growth factor, however, had effects similar to those of EGF and TRH. Of five pituitary cell strains tested, all but one responded to chronic EGF treatment with specifically altered hormone production. Acute chronic EGF treatment with specifically altered hormone production. Acute treatment (30 min) of GH(4)C(1) cells with 10(-8) M EGF caused a 30 percent enhancement of prolactin release compared to a greater than twofold increase caused by 10(-7) M TRH. Therefore, although EGF and TRH have qualitatively similar effects on GH(4)C(1) cells, their powers to affect hormone release acutely or hormone synthesis and cell proliferation chronically are distinct.

Inhibition of biological activity of multiplication-stimulating activity by binding to its carrier protein.

Abstract: Multiplication-stimulating activity (MSA) produced by Buffalo rat liver cells (BRL-3A) in culture is related to the somatomedin family of growth regulatory polypeptides MSA will stimulate glucose transport and DNA synthesis in normal chicken embryo fibroblasts (CEF) at concentrations of 10-200 ng/ml MSA found in BRL-3A-conditioned medium, like the somatomedins in serum, does not exist as the free hormone but is bound to a specific high molecular weight carrier protein In this report we demonstrate that purified MSA carrier protein (MCP) inhibits the biological activity of MSA on CEF as measured by the stimulation of glucose transport and DNA synthesis In addition, purified MCP competitively inhibits the binding of 125I-labeled MSA to these cells In control experiments in which insulin was used as the mitogenic agent, MCP had no effect on these biological responses These results indicate that the inhibitory effect of MCP is the result of specific interaction with MSA and support the hypothesis that cells may be unresponsive to somatomedins bound to their serum carrier proteins

Genes which control cell proliferation in the yeast Saccharomyces cerevisiae

Abstract: In many eukaryotes it is thought that cell proliferation is regulated at a point in G1 close to the initiation of DNA synthesis1. Hartwell2,3 and his colleagues have shown such a point in G1 phase in the budding yeast, Saccharomyces cerevisiae, defined by the cdc 28 mutation. He has termed this point ‘start’ and showed that for cells to proceed beyond start, initiate DNA synthesis and produce a bud, various conditions must be met. Two of these conditions are the presence of adequate nutrients in the medium and the attainment of a critical size. We identify here some of the genes controlling start by isolating mutants which are altered with respect to the conditions in which start occurs. Two types of mutant have been isolated. One results in bud initiation when the parent cell is only half the size at which bud initiation occurs in wild-type cells. Such mutants define a single gene, whi-1, and they are apparently analogous to the size mutants isolated by Nurse and his colleagues4–6 in Schizosaccharomyces pombe. A second type of mutation affects a second gene, whi-2, which is involved in the mechanism whereby cells arrest in G1 in stationary phase. whi-2− cells growing exponentially initiate buds at the same size as wild-type cells. In stationary phase, however, whi-2− cells, unlike wild-type cells, are predominantly budded and are smaller than wild-type cells.

Human cell-surface glycoprotein with unusual properties.

Abstract: We report here the identification of human cell-surface glycoprotein with unusual and interesting properties. We initially detected this glycoprotein on the surface of cultured human haematopoietic cell lines by means of a monoclonal antibody. Although it is expressed on all cultured human haematopoietic cell lines tested, including the inducible promyelocytic tumour cell line HL-60 (ref. 1), it is not present in readily detectable amounts on most normal or leukaemic human haematopoietic cells. HL-60 cells, on exposure in vitro to appropriate chemical inducers, undergo morphological and functional differentiation along the granulocytic pathway or into macrophage-like cells. One consequence of in vitro induction in the specific loss of the glycoprotein from the surface of HL-60 cells. The molecule does not, however, seem to be a highly tissue-specific differentiation antigen because it is also found on human tumour cell lines derived from non-haematopoietic tissues. Rather, its expression seems to be related to cell proliferation. Preliminary chemical characterization suggests that the glycoprotein maybe identical to the abnormal glycoprotein previously reported by Bramwell and Harris to be associated with malignancy.

Mechanism of Action of Antifungal Drugs, with Special Reference to the Imidazole Derivatives

Abstract: Currently used antifungal drugs are distinct in terms of spectrum of activity, potency, therapeutic index, development of resistance, and mode of use. An important factor in the usefulnesss of a compound is the mechanism by which it attacks the structure and function of the fungal cell. The target organelles have been established for most antifungal drugs. Polyenes bind irreversibly to cell membranes. Alteration of the permeability of these structures precedes metabolic disruption and cell death. Griseofulvin deteriorates spindle and cytoplasmic microtubules, influencing cell division and outgrowth of hyphal tips. Flucytosine is deaminated to 5-fluorouracil, which is then phosphorylated and incorporated into RNA; protein synthesis is consequently impaired. A mechanism of action via inhibition of DNA synthesis is an alternative explanation. The imidazole derivatives inhibit the biosynthesis of ergosterol, the main sterol in membranes of fungi. These agents also affect the synthesis of triglycerides and phospholipids. Changes in oxidative and peroxidative enzyme activities, leading to an intracellular buildup of toxic concentrations of hydrogen peroxide, may contribute to the observed deterioration of subcellular organelles and to cell necrosis. The imidazole derivatives inhibit the transformation of blastospores of Candida albicans into the invasive mycelial form. This inhibition probably facilitates the task of host defense cells and may be the principal factor leading to clearance of infection.

Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response.

Abstract: Cultured rat pituitary tumor cells, GH3/D6, which synthesize both growth hormone and prolactin, have cell-surface epidermal growth factor (EGF) receptor sites (34,000 per cell) that bind 125I-labeled EGF with a high affinity (Kd approximately 1 nM). Prolonged treatment of the cells with EGF did not stimulate cell division but did inhibit thyroid hormone-stimulated cell growth. In addition, EGF altered the morphology of the cells from a rounded to an elongated conformation. EGF also induced a perturbation of chromatin structure in GH3 cell nuclei that was detected by an increase (40%) in the number of rifampicin-resistant initiation sites for bacterial RNA polymerase. This was accompanied by an increased synthesis of prolactin and an inhibition of synthesis of growth hormone. In the presence of EGF, the synthesis of growth hormone was no longer inducible by thyroid hormone, but it remained responsive to glucocorticoids. The results demonstrate that EGF can elicit major effects on the cellular phenotype and expression of specific genes in the absence of a proliferative response. This suggests that EGF can also regulate differentiated cellular functions.

Monovalent ion fluxes and the control of cell proliferation in cultured fibroblasts

Abstract: Normal, untransformed fibroblasts reduce their rate of entry into the S (DNA synthesizing) phase of the cell cycle and accumulate in a highly viable state (called Go, or A or R) under a large number of non-optimal environmental condition^.'^ Under usual culture conditions the limiting component is the concentration of serum present in the Addition of serum to such quiescent cultures enhances the rates of protein and RNA synthesis and dramatically stimulates DNA synthesis and cell division. This large and reproducible transition of growth rate provides a useful experimental system for elucidating mechanisms of growth control. Furthermore, quiescent cultures of 3T3 and 3T6 fibroblasts can be stimulated to enter S phase by peptides added at low concentration in serum-free medium.’. ’ * A central problem in understanding the mechanism of action of growth-promoting factors is to determine how, after binding to specific surface receptors, such factors elicit metabolic responses in the cell. A number of models have been proposed to explain the molecular basis of the signallns mechanism invoived in the action of growth factors. These models are based either on the premise that fluctuations in the cellular concentration of a coordinating molecule or ion modulate all other metabolic changes linked to cell proliferation’-”. ” or on possible changes in the spatial distribution of receptors and effectors in the plane of the cell membrane.lO~ ’’Cyclic nucleotides and divalent cations have received considerable attention as possible “second messengers” in the action of growth-promoting factors, but their role remains uncertain. In the present paper we will present an alternative or complementary possibility, namely that changes in monovalent ion fluxes and concentration might play an important role in mediating some aspects of the proliferative response elicited by serum, growth-stimulating hormones and tumor promotors in quiescent cultures of fibroblastic cells.

Localized secretion of acid phosphatase reflects the pattern of cell surface growth in saccharomyces cerevisiae

Abstract: Secretion of cell wall-bound acid phosphatase by Saccharomyces cerevisiae occurs along a restricted portion of the cell surface. Acid phosphatase activity produced during derepressed synthesis on a phosphate-limited growth medium is detected with an enzyme-specific stain and is localized initially to the bud portion of a dividing cell. After two to three generations of phosphate-limited growth, most of the cells can be stained; if further phosphatase synthesis is repressed by growth in excess phosphate, dividing cells are produced in which the parent but not the bud can be stained. Budding growth is interrupted in alpha-mating-type cells by a pheromone (alpha-factor) secreted by the opposite mating type; cell surface growth continues in the presence of alpha-factor and produces a characteristic cell tip. When acid phosphatase synthesis is initiated during alpha-factor treatment, only the cell tip can br stained; when phosphate synthesis is repressed during alpha-factor treatment, the cell body but not the tip can be stained. A mixture of derepressed alpha cells and phosphatase-negative alpha cells form zygotes in which mainly one parent cell surface can be stained. The cell cycle mutant, cdc 24 (Hartwell, L.H. 1971. Exp. Cell Res. 69:265-276), fails to bud and, instead, expands symmetrically as a sphere at a nonpermissive temperature (37 degrees C). This mutant does not form a cell tip during alpha-factor treatment at 37 degrees C, and although acid phosphatade secretion occurs at this temperature, it is not localized. These results suggest that secretion reflects a polar mode of yeast cell- surface growth, and that this organization requires the cdc 24 gene product.

Constitutive uncoupling of pathways of gene expression that control growth and differentiation in myeloid leukemia: a model for the origin and progression of malignancy.

Abstract: Chemical carcinogens and tumor promoters have pleiotropic effects. Tumor initiators can produce a variety of mutations and tumor promotres can regulate a variety of physiological molecles that control growth and differentiation. The appropriate mutation and the regulation of the appropriate molecules to induce cell growth can initiate and promote the sequence of changes required for transformation of normal cells into malignant cells. After this sequence of changes, some tumors can still be induced to revert with a high frequency from a malignant phenotype to a nonmalignant phenotype. Results obtained from analysis of regulation of growth and differentiation in normal and leukemic myeloid cells, the phenotypic reversion of malignancy by induction of normal differentiation in myeloid leukemia, and the blocks in differentiation-defective leukemic cell mutants have been used to propose a general model for the origin and progression of malignancy. The model states that malignancy originates by changing specific pathways of gene expresion required for growth from inducible to constitutive in cells that can still be induced to differentiate normally by the physiological inducer of differentiation. The malignant cells, unlike the normal cells, then no longer require the physiological inducer for growth. This changes the requirements for growth and uncouples growth from differentiation. Constitutive expression of other specific pathways can uncouple other controls, which then causes blocks in differentiation and the further progression of malignancy. The existence of specific constitutive pathways of gene expression that uncouple controls in malignant cells can also exlain the expresion of fetal proteins, hormones, and some other specialized products of normal development in various types of tumors.

Interferon effects on microfilament organization, cellular fibronectin distribution, and cell motility in human fibroblasts.

Abstract: We have shown previously (Pfeffer et al., 1979, Exp. Cell Res. 121:111-120) that treatment of human fibroblasts, planted at a density of 2x10(3) cells/cm(2), with purified human fibroblasts interferon (640 U/ml) for 3 d at 37 degrees C decreases the overall rate of cell proliferation to 35-40 percent of the control value. In the present experiments we have characterized the phenotype of interferon-inhibited fibroblasts. The mean volume of trypsinized, interferon-treated cells was increased 31 percent abover that of control cells. The interferon-treated population was much more heterogeneous than the control population with respect to volume, and there was a considerable overlap in the volume distributions of the two populations. The cell surface area was, on the average, increased 65 percent after interferon treatment. More than 80 percent of the treated cells had enlarged nuclei, many of which were lobed, and the fraction of binucleated cells was increased fivefold. After interferon treatment, over 40 percent of the cells showed large actin-containing fibers in the form of multiple parallel arrays. Fewer than 5 percent of the control cells contained such large actin fibers. The number of actin fibers of all sizes was tripled in the treated fibroblasts on a per cell basis and, calculated per unit surface area of the cells, the number was increased 82 percent. In contrast, 10-nm filaments and microtubules did not appear to be increased in number per unit surface area of the cells. The increases per cell in the abundance of these structures were directly related to increased cell size. After interferon treatment, fibronection was distributed in arrays of long filaments covering most portions of the cell surface. Interferon treatment markedly decreased the rate of cell locomotion as well as membrane ruffling and saltatory movements of intracellular granules.

Role of G0-G1 arrest in the inhibition of tumor cell growth by interferon.

Abstract: We report here that human leukocyte interferon preparations are capable of influencing the transition of human melanoma cells from the "A" state to the "B" phase Human melanoma cells that enter a quiescent stage at high cell density are more sensitive to the cytostatic action of interferon than those that continue to proliferate under similar conditions Cell cycle perturbations caused by interferon in these cells include a decreased transition rate out of G0-G1 ("A" state) into S ("B" phase) and a prolongation of S These findings support the idea that some metabolic event is required for progress through the G0-G1 phase of the cell cycle and is susceptible to interferon action

Evidence for the Cytotoxic Activity of Polyglutamate Derivatives of Methotrexate

Abstract: The potential role of the polyglutamate derivatives of methotrexate in the cytotoxicity of methotrexate has been examined in H-35 hepatoma cells in culture. Pulse doses of methotrexate result in the accumulation of a nonexchangeable fraction of methotrexate that is toxic to the cells and consists almost entirely of polyglutamates. The toxicity of the polyglutamates appears to correlate with their effects on de novo thymidine synthesis, which was measured in intact cells by the release of tritium from [5-3H]deoxyuridine. Evidence for this comes from the observation that the dose-inhibition response following pulses of methotrexate is nearly identical for cell growth and tritium release. Protection studies demonstrated that both thymidine and hypoxanthine are needed to prevent methotrexate toxicity, offering evidence that inhibition of cell growth is due to a depletion of reduced folate coenzymes. Cells treated with methotrexate were analyzed for the composition of the species bound to the target enzyme dihydrofolate reductase (EC 1.5.1.3). With 0.03 and 10 µM methotrexate in the medium the dihydrofolate reductase bound material consisted of 82 and 95% polyglutamates, respectively, compared with 78 and 88% in the total cell pool. These results demonstrate that the polyglutamates have at least an equivalent affinity for the enzyme in the intact cell when compared to methotrexate and, as such, can be chiefly responsible for the toxicity of methotrexate in those cells that have sufficient capacity to convert methotrexate to its γ-linked glutamate derivatives.

Growth of Human Small Cell (Oat Cell) Carcinoma of the Lung in Serum-free Growth Factor-Supplemented Medium

Abstract: A cell culture line of human small cell (oat cell) carcinoma of the lung (NCI-H69) was studied for growth factor requirements for replication in serum-free medium. By a series of addition experiments, the combination of selenium (3 × 10 -8 m), hydrocortisone (10 -8 m), insulin (5 µg/ml), transferrin (100 µg/ml), and 17β-estradiol (10 -8 m) (SHITE) added to Roswell Park Memorial Institute Medium 1640 without serum was found to allow optimum replication when cells were transferred from serum-containing medium. NCI-H69 cells have replicated continuously in SHITE-supplemented Roswell Park Memorial Institute Medium 1640 for periods of more than 12 months. The cells replicate with approximately the same doubling times as in medium supplemented with 10% fetal calf serum, but they exhibited a lower saturation density and longer lag phase in SHITE-supplemented medium compared to serum-supplemented medium. When individual components of SHITE were deleted, immediate and dramatic differences in growth were seen only with deletion of transferrin while minimal decreases were noted with deletion of the other factors. After 6 months of growth in SHITE, deletion of insulin or transferrin from the formula resulted in cessation of cell growth in serum-free medium while deletion of hydrocortisone or estradiol (alone or together) did not. A variety of other hormones and growth factors did not add significantly to SHITE in promoting replication. In addition, a series of hormones was found to inhibit replication or saturation density achieved in serum-free, hormone-supplemented medium including epidermal growth factor, luteinizing hormone-releasing factor, parathyroid hormone, blood meal, and the tripeptide glycyl-l-histidyl-l-lysine acetate. Six other small cell carcinoma lines were tested and also were able to replicate in SHITE-supplemented serum-free medium indicating the generality of this formula for small cell carcinoma, NCI-H69 cells cultured for 4 months in SHITE medium retained their amine precursor uptake and decarboxylation properties, characteristic small cell carcinoma histology by light microscopy, dense core (neurosecretory) granules by electron microscopy, and tumorigenicity in nude mice. The identification of this growth formula should allow selection of tumor cells directly from patient samples, study of hormone production and regulation by these cell lines, and identification of new growth and therapeutic agents, and it should provide a way of selecting for normal amine handling cells related histogenetically to small cell carcinoma.

In Vitro Inhibition of Cell Growth, Collagen Synthesis, and Prolyl Hydroxylase Activity by Triamcinolone Acetonide

Abstract: SummaryThe effects of triamcinolone acetonide on fibroblasts isolated from normal dermis and keloids were tested by measuring cell division, DNA content, collagen synthesis, and prolyl hydroxylase activity following steroid treatment. In the present study, the DNA content and cell division of normal fibroblasts were inhibited by 50 μg/ml steroid to a greater extent than keloid fibroblasts. Collagen synthesis was inhibited by triamcinolone acetonide in a similar, dose-dependent manner in both keloid-derived and normal skin fibroblasts. Although triamcinolone acetonide decreased prolyl hydroxylase in all cultures, this inhibition did not correlate with the steroid-induced inhibition of collagen synthesis. In addition, triamcinolone acetonide increased the specific activity of radioactive proline in the amino acid cell pool 37–38% in all cultures. Triamcinolone acetonide enhanced TCA-soluble, [14C]hydroxyproline production in both cell types, suggesting that collagen degradation was increased by this cortico...

Induction of supernumerary tracheal buds and the stimulation of DNA synthesis in the embryonic chick lung and trachea by epidermal growth factor.

Abstract: Epidermal growth factor (EGF) has been found to stimulale DNA synthesis in both the trachea and bronchial tree of 5-day-old chick embryo lung rudiments in organ culture. After 20 h culture in the presence of 10 ng/ml EGF, the incorporation of tritiated thymidine into DNA is stimulated two- to three-fold following a 2 h labelling period, as revealed by scintillation counting. Autoradiographic data indicate that this stimulation is most marked in the epithelial tissue component of both the trachea and bronchial tree. Supernumerary ‘lung’ buds have been induced in the normally unbranched tracheal epithelium by agarose pellets containing EGF, such buds having been previously induced only by grafting a variety of mesenchymal tissues alongside the tracheal epithelium. Since EGF has been shown to be a potent stimulator of tracheal DNA synthetic activity it is suggested that the induction of supernumerary buds by the EGF-agarose pellets is achieved through a localized stimulation of cell proliferation in the tracheal epithelium. These data would further suggest that the induction of supernumerary tracheal buds by various mesenchymal tissues is similarly due to a localized increase in mitotic activity resulting from the action of some mitotic stimulator substance(s) emanating from the inducing mesenchymal tissue. This conclusion may be extended to include normal bud formation which occurs during branching morphogenesis in everal developing organ systems.

Stimulation of DNA synthesis in quiescent cultured cells: exogenous agents, internal signals, and early events.

Abstract: Publisher Summary Animal cells in vivo exist in a nonproliferating state in which they remain viable and metabolically active They arose from proliferating cells whose metabolic patterns were switched to “quiescence” in G 1 –G o at some time during differentiation However, the cells of many tissues and organs retain the capacity to respond to extracellular signals such as hormones, peptide factors, and antigens by increasing the rate of cell proliferation Various aspects of growth control can be studied in cell culture and can be shown to correlate with such in vivo counterparts as tumorigenicity A manifestation of growth control in cell culture is seen in the ability of normal, untransformed fibroblasts to reduce their rate of entry into the DNA-synthesizing phase of the cell cycle and to accumulate in a viable state under a large number of nonoptimal environmental conditions This property is evident in cultures of 3T3 cells, a mouse cell line selected for cessation of DNA synthesis at confluence and against the growth of spontaneous variants under crowded conditions Under usual culture conditions, the limiting component is the concentration of serum present in the medium

Amiloride inhibits murine erythroleukemia cell differentiation: evidence for a Ca2+ requirement for commitment

Abstract: The effect of amiloride (an inhibitor of passive Na+ transport in many tissues) on the differentiation of murine erythroleukemia cells was investigated. Amiloride completely blocked the dimethyl sulfoxide (Me2SO)-induced erythroid differentiation of cells at a concentration (10 microgram/ml) that did not affect cell proliferation. Amiloride also prevented the decrease in cell volume normally observed afte a 20-hr exposure to Me2SO. The ratio of total cell Na+ to total cell water was essentially the same for control cells, Me2SO-treated cells, and cells treated with Me2SO plus amiloride. However, cells treated for 24 hr with Me2SO had a rate of Ca2+ uptake that was twice that of untreated cells and a similarly higher Ca2+ content. Addition of amiloride plus Me2SO prevented both the increase in Ca2+ uptake rate and the increase in Ca2+ content. Cells grown in the presence of Me2SO plus amiloride initiated differentiation immediately after removal of amiloride or addition of the Ca2+ ionophore A23187 (1 microgram/ml). Addition of sufficient ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to reduce free extracellular Ca2+ to submicromolar levels prevented Me2SO-induced differentiation while only slightly affecting cell proliferation. These results suggest that an increase in in the Ca2+ level is an essential step in Me2SO induction, that amiloride either directly or indirectly inhibits this process, and that Me2SO has an early effect on cells that is necessary for differentiation and is not mimicked by A23187.