bluuebunny/crossref_metadata_2025 huggingface dataset:
Both normal and tumor cells produce basic fibroblast growth factor
Authors: Moscatelli David , Presta Marco , Joseph‐Silverstein Jacquelyn , Rifkin Daniel B.
Published: February 2005

We have previously purified from human placenta a basic fibroblast growth factor (FGF)‐like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial (BCE) cells, and induces angiogenesis in vivo. The ability of basic FGF to stimulate PA production in BCE cells was used as an assay for the presence of basic FGF‐like molecules in extracts of both normal and tumor‐derived cultured cells. The identity of the PA‐stimulatory activity with basic FGF was confirmed by its high affinity for heparin and by its cross‐reactivity with antibodies to human placental basic FGF. Basic FGF‐like molecules were identified in eight of ten cell lines tested, and the amount of FGF‐like activity present in these cells bore no relation to their origin from normal or tumor tissue. The test cells, BCE cells, had one of the highest levels of FGF‐like activity, suggesting that it may have an autocrine role in these cells.
Isolation and characterization of mink lung epithelial cell mutants resistant to transforming growth factor β
Authors: Chinkers Michael
Published: February 2005

Mink lung epithelial cells resistant to growth inhibition by transforming growth factor β (TGF‐β) have been isolated by chemical mutagenesis and growth in the presence of platelet extracts enriched in TGF‐β. Several resistant clones were isolated, at least one of which stably retained its resistance to TGF‐β when grown in the absence of the factor. The cells of this clone were similar to the parent cells in morphology and growth properties. However, unlike the parent cells, the resistant cells did not show any of the following responses to TGF‐β: (1) inhibition of DNA synthesis and proliferation; (2) morphological changes involving increased cell spreading; or (3) stimulation of synthesis of a 48‐kilodalton secreted protein. The resistant cells do, however, retain a functional TGF‐β receptor. The TGF‐β resistant cell lines may be useful in genetic studies designed to identify the biochemical events required for inhibition of epithelial cell growth by this factor.
Peptide inhibitors of fibronectin, laminin, and other adhesion molecules: Unique and shared features
Authors: Yamada Kenneth M. , Kennedy Dorothy W.
Published: February 2005

Synthetic peptides can specifically inhibit the function of certain adhesive glycoproteins in vitro and in vivo. We have compared the relative activities of a set of six variant synthetic peptides based on the sequence of fibronectin in terms of their ability to inhibit the interactions of fibroblasts with fibronectin, spreading factor/vitronectin, laminin, and native collagen gels. BHK (baby hamster kidney) and chick embryo fibroblasts spreading on these adhesive molecules displayed distinctive patterns of sensitivity to inhibition by this panel of peptides, which depended on the adhesive molecule rather than the cell type. For fibronectin, Gly‐Arg‐Gly‐Asp‐Ser was considerably more active than Arg‐Gly‐Asp‐Ser, whereas these two peptides displayed little difference in activity in inhibiting cell adhesion to spreading factor. For both proteins, the inverted peptide sequence Ser‐Asp‐Gly‐Arg was also moderately active, whereas closely related peptides containing a transposition, a deletion, or a single, conserved amino acid substitution were much less active. For inhibiting interactions with laminin or native type I collagen gels, Gly‐Arg‐Gly‐Asp‐Ser was only weakly active, but the inverted peptide Ser‐Asp‐Gly‐Arg unexpectedly continued to display inhibitory activity for both attachment proteins in both cell types. Our results indicate that different adhesive processes depend on distinct peptide recognition events by a cell. However, there may be a possible common denominator among attachment proteins in a moderate sensitivity to Ser‐Asp‐Gly‐Arg. Our study also underscores the importance of examining a full set of peptide analogs when these novel inhibitors are used to characterize biological processes.
Soluble antigen induces T lymphocytes to secrete an endoglycosidase that degrades the heparan sulfate moiety of subendothelial extracellular matrix
Authors: Fridman Rafael , Lider Ofer , Naparstek Yaakov , Fuks Zvi , Vlodavsky Israel , Cohen Irun R.
Published: February 2005

The antigen‐mediated induction of heparanase, an endoglycosidase capable of degrading heparan sulfate from the subendothelial extracellular matrix (ECM), was investigated in a rat T lymphocyte cell line reactive against the basic protein (BP) of myelin. We found that nonactivated T lymphocytes could be induced to express heparanase activity following exposure to soluble but not to ECM‐bound BP. The induction of heparanase was immunologically specific and indpendent of the presence of syngeneic or allogeneic antigen presenting cells (APC). However, anti‐IA antibodies inhibited heparanase expression. Soluble BP induced secretion of heparanase into the culture medium within minutes, despite inhibition of protein synthesis. Cell lysates of T lymphocytes contained heparanase activity. Thus, T lymphocytes secrete a preformed heparanase following exposure to specific antigen.
Propagation of differentiating normal human tracheobronchial epithelial cells in serum‐free medium
Authors: Chopra D. P. , Sullivan J. , Wille J. J. , Siddiqui K. M.
Published: February 2005

Serial‐passage cultures of normal human tracheobronchial (TB) epithelial cells that exhibit functional differentiation have been established in serum‐free medium supplemented with bovine pituitary extract (25 μg/ml), insulin (5 μg/ml), hydrocortisone (0.5 μg/ml), EGF (5 ng/ml), 10−6M each of ethanolamine and phoshoethanolamine, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 80 hours. Further, the passaged cultures retained differentiated morphology as evidenced by secretion of glycoproteins, binding of concanavalin A lectin, and presence of alcian blue and periodic acid Schiff‐positive material in their cytoplasm. Ultrastructural observations further supported the functional epithelial nature of the cultures. Most cells exhibited characteristic microvilli on cell surfaces and showed junctional complexes between them. The cytoplasm contained a large number of perinuclear secretory vesicles, a characteristic feature of the differentiated cells. These cultures provide an excellent model to study factors that regulate synthesis and secretion of glycoproteins in normal human TB cells.
Deoxyribonucleotide metabolism and cyclic AMP resistance in hydroxyurea‐resistant S49 T‐lymphoma cells
Authors: Albert Daniel A. , Gudas Lorraine J. , Nodzenski Edwardine
Published: February 2005

We investigated the cell cycle regulation of deoxyribonucleoside triphosphate (dNTP) metabolism in hydroxyurea‐resistant (HYUR) murine S49 T‐lymphoma cell lines. Cell lines 10‐ to 40‐fold more hydroxyurea‐resistant were selected in a stepwise manner. These HYUR cells exhibited increased CDP reductase activity (5‐ to 8‐fold) and increased dNTP pools (up to 5‐fold) that appeared to result from increased activity of the M2 subunit (binding site of hydroxyurea) of ribonucleotide reductase. These characteristics remained stable when the cells were grown in the absence of hydroxyurea for up to 2 years. In both wild type and hydroxyurea‐resistant cell populations synchronized by elutriation, dCTP and dTTP pools Increased in S phase, whereas dATP and dGTP pools generally remained the same or decreased, suggesting that allosteric effector mechanisms were operating to regulate pool sizes. Additionally, CDP reductase activity measured in permeabilized cells increased in S phase in both wild type and hydroxyurea‐resistant cells, suggesting a nonallosteric mechanism of increased ribonucleotide reductase activity during periods of active DNA synthesis. While wild type S49 cells could be arrested in the G1 phase of the cell cycle by dibutyryl cyclic AMP, hydroxyurea‐resistant cell lines could not be arrested in the G1 phase by exogenous cyclic AMP or agents that elevate the concentration of endogenous cyclic AMP. These data suggest that cyclic AMP‐generated G1 arrest in S49 cells might be mediated by the M2 subunit of ribonucleotide reductase.
Phorbol esters induce angiogenesis in vitro from large‐vessel endothelial cells
Authors: Montesano R. , Orci L.
Published: February 2005

We have shown previously that the tumor promoter phorbol myristate acetate (PMA) induces capillary endothelial cells grown on the surface of threedimensional collagen gels to invade the underlying matrix as capillary‐like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell 42:469, 1985). To establish whether the potential to invade the extracellular matrix as capillary‐like sprouts is restricted to microvascular endothelial cells or is also shared by large vessel endothelium, we have examined the response to PMA of endothelial cells isolated from the human umbilical vein and the calf pulmonary artery. The results of these experiments show that both types of macrovascular endothelial cells are able to penetrate into collagen gels as vessel‐like tubes following treatment with PMA. This demonstrates that endothelial cells derived from large vessels can, in response to appropriate signals, express invasive properties thought to be associated specifically with capillary endothelial cells in vivo.
Suppression of maturation of megakaryocyte colony forming unit in vitro by a platelet‐released glycoprotein
Authors: Dessypris Emmanuel N. , Gleaton John H. , Sawyer Stephen T. , Armstrong Otho L.
Published: February 2005

The suppressive role of platelets on the grwoth of human marrow megakaryocyte colony forming units (CFU‐M) in vitro was investigated by the use of a plasma clot assay. An inverse correlation was established between the number of megakaryocytic colonies grown and the platelet concentration of the plasma or the resultant serum used in the culture system. The suppressive effect of platelets on megakaryocyte colony formation reached a plateau at normal human blood platelet concentration and was specific for CFU‐M growth, since marrow cell erythroid burst formation (BFU‐E) and granulocytic‐monocytic colony formation (CFU‐GM) remained unaffected. The inhibitory activity was detectable in the supernatants of platelet suspensions aggregated by thrombin or ADP, and the inhibitory activity released from ADP‐stimulated platelets was blocked by pretreatment of platelets with monoclonal antibody HuPl‐m1. Partial purification of this activity was acheived by diethylaminoethyl (DEAE)‐ion exchange and phytohemagglutinin (PHA)‐E agarose affinity chromatography. This inhibitor is a glycoprotein with a molecular weight of 12‐17K daltons. This platelet released glycoprotein does not affect the early proliferative phase of CFU‐M in vitro but acts on a day 6–8 CFU‐M‐derived cell by adversely affecting its maturation into recognizable megakaryocytes. These findings demonstrate that a glycoprotein released from platelets suppresses the maturation of CFU‐M into megakaryocytes.
Intracellular binding of spin‐labeled amiloride: An alternative explanation for amiloride's effects at high concentration
Authors: Costa Charles J. , Kirschner Leonard B. , Cragoe Edward J.
Published: February 2005

Amiloride, an important inhibitor of Na+ transport and Na+/H+ exchange, has been used in nontransporting tissues to investigate the relationship between ionic fluxes or intracellular pH change and proliferative or synthetic events. Reports that amiloride is permeant and had direct effects on intracellular processes have led us to investigate the possibility that amiloride binds intracellularly to nuclei, mitochondria, and to purified nucleic acids. Using a nitroxide spin‐labeled derivative of amiloride (ASp) and electron paramagnetic responance (EPR) spectroscopy, we have demonstrated that nuclei and mitochondria isolated from trout liver bind significant amounts of ASp especially at the high amiloride concentrations (∼ mM) commonly used to inhibit proliferative events. While the chemical component responsible for ASp binding in these organelles was not identified, native DNA binds significant amounts of ASp whereas single stranded DNA and RNA bind much less. When these observations are taken together with reports of amiloride's direct action on cellular processes, they support the possibility that some of the effects attributed to inhibition of a transport event are caused by amiloride directly.
Modulation by the src oncogene of the effect of inhibitory diffusible factor IDF45
Authors: Blat C. , Villaudy J. , Rouillard D. , Golde A. , Harel L.
Published: February 2005

Density‐dependent inhibition of growth has been assumed to be under the control of inhibitory molecules diffusing from dense cell cultures. Growth inhibitory factors have been fractionated or purified from medium conditioned by different cell types. In the present work, it was shown that IDF45 (inhibitory factor diffusing from 3T3 cells) decreased DNA synthesis in chick embryo fibroblats (CEF) and was an inhibitor of CEF growth; this inhibition was reversible.

Since similitudes between oncogene products and growth factors have been observed, it was of interest to compare the inhibitory effect of IDF45 upon the stimulation of DNA synthesis induced either by serum or by pp60‐src. CEF infected by Ny68 virus (a mutant of Rous sarcoma virus ts for the expression of transformation) were density‐inhibited at 41°C, but were stimulated at this temperature by addition of 1% serum. This stimulation was 94% inhibited by IDF45. The same Ny68‐infected cells could also be stimulated transfer to 37°C, the permissive temperature (in the absence of serum). The stimulation of DNA synthesis by src expression was poorly inhibited by IDF45. From our results, it appears that oncogene expression in CEF induces a loss in their sensitivity to IDF45. This would explain why transformed cells escape DDI of growth.
Insulin binding and processing by H4IIEC3 hepatoma cells: Ultrastructural and biochemical evidence for a unique route of internalization and processing
Authors: Smith Robert M. , Laudenslager Nan H. , Shah Neelima , Jarett Leonard
Published: February 2005

Biochemical and ultrastructural studies of insulin binding and cellular processing by cultured H4IIEC3 hepatoma cells were performed. Insulin binding and intracellular accumulation were rapid and after 30 min at 37°C, 65% of the total cell‐associated 125I‐insulin was in an acid‐stable compartment. Chloroquine had no significant effect on the amount of total cell‐associated insulin or the percentage of insulin in the acid‐stable compartment or cell‐associated insulin degradation under those conditions, but after 60‐min incubations, it slightly decreased the rate of dissociation of internalized hormone. Ultrastructural analysis revealed that monomeric ferritin‐insulin (Fm‐I) initially bound to single or paired receptors on microvilli. Within 5 min occupied insulin receptors microaggregated and migrated to the intervillous cell surface. During the next 5‐10 min occupied receptors aggregated into large clusters on the plasma membrane. Large amounts of insulin were internalized by macropinocytosis and the majority of internalized Fm‐I was found in phagosomes. Less than 10% of the membrane‐bound insulin was associated with pinocytotic invaginations or coated pits and less than 5% of the total cell‐associated insulin was found in lysosomes. Chloroquine had no detectable effect on the amount of Fm‐I or its distribution among the intracellular organelles. These studies demonstrated that, compared to previous studies with rat adipocytes or 3T3‐L1 adipocytes, insulin interalization and intracellular processing in this hepatoma cell were unique. These differences provide further evidence that insulin binding and processing may be controlled by cell‐specific mechanisms and that substantial heterogeneity exists in pathways previously presumed to be similar for all cell types.
Release of angiotensin I‐converting enzyme by endothelial cells in vitro
Authors: Noveral James P. , Mueller Stephen N. , Levine Elliot M.
Published: February 2005

Bovine fetal aortic endothelial cells cultured in serum‐containing medium accumulate angiotensin I‐converting enzyme (ACE) activity and also release it into the culture medium. Following subcultivation of a confluent culture using trypsin‐EDTA, cellular ACE activity falls 50% within 8 h, but no ACE activity is detected in the medium, suggesting intracellular loss of the enzyme activity. ACE activity reappears in both the cell lysate and culture medium after the culture becomes confluent. The rate of accumulation of ACE activity released into the medium is always greater than that for cellular activity. For example, 21 days following subcultivation 80–85% of the total culture activity is detected in the medium. Both cellular and medium‐associated ACE decrease proportionately as the culture progresses through its in vitro lifespan.
Effects of diverse intracellular thiol delivery agents on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione, and ornithine decarboxylase induction in isolated mouse epidermal cells treated with 12‐O‐tetradecanoylphorbol‐13‐acetate
Authors: Perchellet Elisabeth M. , Maatta Eric A. , Abney Nancy L. , Perchellet Jean‐Pierre
Published: February 2005

Since the enhancement of the activity of the natural glutathione (GSH)‐dependent antioxidant protective system of the epidermal cells appears to inhibit the oxidative challenge presumably linked to skin tumor promotion by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), we have compared the effectiveness of diverse intraceliular thiol delivery agents as inhibitors of the effects of TPA on GSH metabolism and ornithine decarboxylase (ODC; L‐ornithine carboxy‐lyase, EC 4.1.1.17) induction in isolated mouse epidermal cells. Here we report that, at a 2‐mM concentration, the monoethyl and monomethyl esters of GSH, N‐acetyl‐L‐cysteine, and L‐2‐oxothiazolidine‐4‐carboxylate are all significantly more effective than GSH in inhibiting the sharp decline in the intracellular ratio of reduced GSH/oxidized glutathione (GSSG), the prolonged decrease in GSH peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity, and the induction of ODC activity caused by 1 μM TPA. Moreover, diethyldithiocarbamate prevents totally the initial drop in the GSH/GSSG ratio of TPA‐treated cells and is the most potent inhibitor of TPA‐decreased GSH peroxidase activity in relation with its remarkable 98% inhibition of TPA‐induced ODC activity, suggesting that the potential antitumor‐promoting activity of this compound in mouse skin may be far superior to that previously demonstrated by GSH in the initiation‐promotion protocol.
High and low affinity binding sites for basic fibroblast growth factor on cultured cells: Absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells
Authors: Moscatelli David
Published: February 2005

Scatchard analysis of binding of 125l‐basic fibroblast growth factor (FGF) to baby hamster kidney (BHK) cells revealed the presence of two binding sites: a high affinity site with KD of 20 pM and 80,000 sites per cell and a low affinity site with KD of about 2 nM and 600,000 sites per cell. The binding to the two sites could be separated by first washing the cells with 2 M NaCl at pH 7.5 which released the low affinity binding and then extracting the cells with 0.5% Triton X‐100 to recover the 125l‐basic FGF bound to high affinity sites. The binding to the high affinity site was acid sensitive, suggesting that it represented binding to the receptor. Binding to the low affinity site could be competed strongly by heparin and less strongly by heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. Treatment of BHK cells with heparinase abolished 62% of the low affinity binding, suggesting that the low affinity binding represented binding to cell‐associated, heparin‐like molecules. A variety of other cell types, including bovine capillary endothelial (BCE) cells, also demonstrated both low and high affinity binding sites. To test whether the low affinity binding might play a role in the basic FGF stimulation of plasminogen activator (PA) production by BCE cells, heparin was added to BCE cultures at concentrations which totally blocked binding of 125l‐basic FGF to the low affinity sites. Addition of the heparin did not diminish the increased PA production induced by basic FGF. This suggests that the low affinity binding has no direct role in the stimulation of PA production in BCE cells.
Isolation of an autocrine growth factor from hepatoma HTC‐SR cells
Authors: Ove Peter , Coetzee Mona L. , Scalamogna Philip , Francavilla Antonio , Starzl Thomas E.
Published: February 2005

A growth factor has been isolated from HTC‐SR rat hepatoma tissue culture cells which specifically stimulates DNA synthesis and cell proliferation of the HTC cells that produce it. The factor can be isolated from HTC cell conditioned medium or from an HTC cell extract. This autocrine factor has been purified 640‐fold from a postmicrosomal supernatant by successive steps, involving ethanol precipitation, heating at 80°C for 10 min, chromatography on a DEAE Bio‐Gel A column, and chromatography on a heparin‐sepharose affinity column. The major peak of activity eluted from the heparin column migrates as a single band on SDS‐PAGE with an apparent Mr of 60,000. The factor is resistant to acid, heat, and neuraminidase but sensitive to trypsin, papain, and protease.

The autocrine nature of the factor is indicated by the finding that several other types of cells do not respond with increased DNA synthesis. Mouse L‐cells, BHK cells, Novikoff hepatoma cells, hepatocytes in primary culture, and an epithelial‐like rat liver‐derived cell line (Clone 9) were tested, and none of the cells could be stimulated. Small amounts of the factor could be extracted from the Clone 9 cells, however. This material had the same physical and purification properties as the factor extracted from HTC cells, but it did not stimulate DNA synthesis in Clone 9 cells, only in HTC cells.

Addition of the factor resulted in an almost immediate stimulation of DNA synthesis in a proliferating HTC cell population. When the factor was added together with [3H]thymidine for 2 h, a significant stimulation of DNA synthesis was observed, provided the addition was made between 18 and 48 h after the cells had been plated. Autoradiographic studies indicated that the factor both accelerates DNA synthesis in cells already making DNA and increases the number of cells entering the S period. The stimulation of DNA synthesis was completely inhibited by 10 mM hydroxyurea, whether the factor was present for 2, 24, or 48 h in the culture. A significant increase in cell number due to addition of the factor was also observed. This accelerated proliferation was detectable only after the cells had been in culture for at least 48 h with the factor present.
Zinc‐induced reduction in melphalan cytotoxicity: Heterogeneity of response of cloned human tumor cells
Authors: Tobey Robert A. , Tesmer Judith G. , Shackelford Mary E.
Published: February 2005

Previous studies with cultured human normal fibroblasts indicated that pretreatment of the cells with zinc for 12 h prior to exposure to the alkylating agent melphalan increased survival by seven‐ to ninefold over survival values obtained in cultures treated with drug only. Comparable pretreatment of cells derived from a variety of human tumors resulted in an increase in survival of 1.7‐fold or less. To determine whether the limited responsiveness to zinc represented a general property of tumor cells (which would be characterized by a lack of highly zinc‐responsive subpopulations contained within the parental tumor populations), a series of clones was prepared from the A101D human melanoma line and the A549 human alveolar cell carcinoma line. Cells from each clone were then challenged with melphalan with and without zinc pretreatment. Twenty‐five percent of the tumor clones exhibited increased resistance to melphalan following pretreatment with zinc (range of 2.1‐ to 5.2‐fold increase in survival), indicating that the parental tumor lines were highly heterogeneous in regard to inducibility to a state of reduced sensitivity to melphalan. There was no evidence of a relationship between zinc‐induced reductions in toxicity and induced elevations in total intracellular glutathione content, indicating that the primary effect of zinc is not directed toward elevating intracellular levels of glutathione.
Volume‐regulating behavior of human platelets
Authors: Livne Avinoam , Grinstein Sergio , Rothstein Aser
Published: February 2005

Human platelets exposed to hypotonic media undergo an initial swelling followed by shrinking (regulatory volume decrease [RVD]). If the RVD is blocked, the degree of swelling is in accord with osmotic behavior. The cells could swell at least threefold without significant lysis. Two methods were used to follow the volume changes, electronic sizing and turbidometery. Changes in shape produced only limited contribution to the measurements. The RVD was very rapid, essentially complete in 2 to 8 minutes, with a rate proportional to the degree of initial cell swelling. RVD involved a loss of KCI via volume‐activated conductive permeability pathways for K+ and anions, presumably Cl −. In media containing > 50 mM KCI, the shrinking was inhibited and with higher concentrations was reversed (secondary swelling), suggesting that it is driven by the net gradient of K+ plus Cl−. The K+pathway was specific for Rb+ and K+ compared to Li+ and Na+. The Cl− pathway accepted NO3− and SCN− but not citrate or SO2‐4. In isotonic medium, the permeability of platelets to Cl− appeared to be low compared to that of K+. After hypotonic swelling both permeabilities were increased, but the Cl− permeability exceeded that of K+. The Cl− conductive pathway remained open as long as the cells were swollen. RVD was incomplete unless amiloride, an inhibitor of Na+ /H+ exchange, was present or unless Na+ was replaced by an impermeant cation. In addition, acidification of the cytoplasm occurred upon cell swelling. This reduction in pHi appeared to activate Na+/ H+ exchange, with a resultant uptake of Na+ and reduction in the rate and amount of shrinking. Like other cells, platelets responded to hypertonic shrinking with activation of Na+/H+ exchange, but regulatory volume increase was not detectable.
Recombinant murine granulocyte‐macrophage (GM) colony‐stimulating factor supports formation of GM and multipotential blast cell colonies in culture: Comparison with the effects of interleukin‐3
Authors: Koike Kenichi , Ogawa Makio , Ihle James N. , Miyake Takaji , Shimizu Tomoe , Miyajima Atsushi , Yokota Takashi , Arai Ken‐Ichi
Published: February 2005

We studied the effects of murine recombinant granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) on murine hemopoiesis in methylcellulose culture. The GM‐CSF was purified from cultures of Saccharomyces cerevisiae transfected with a cloned murine GM‐CSF cDNA. In cultures of spleen cells from normal mice, only granulocyte‐macrophage (GM) colonies were supported by GM‐CSF. Blast cell colonies were the predominant type in cultures of spleen cells from 5‐fluorouracil (5‐FU)‐treated mice. Dose‐response studies revealed that maximal GM and blast cell colony formation is achieved with 100 U/ml GM‐CSF. Blast cell colonies revealed variable but high replating efficiencies, and the secondary colonies included multilineage colonies. Serial replating of washed blast cell colonies in cultures with GM‐CSF provided evidence for the direct effects of GM‐CSF on the proliferation of multipotential blast cells. A combination of GM‐CSF and interleukin‐3 (IL‐3) did not increase the number of blast cell colonies over the level supported by IL‐3. This observation indicates that the progenitors for blast cell colonies that responded to GM‐CSF are a subpopulation of multipotential progenitors that are supported by IL‐3. Cytological studies of colonies derived from GM‐CSF and/or IL‐3 suggest that the eosinophilopoietic ability of murine GM‐CSF is less than that of IL‐3.
Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells
Authors: Roberts R. A. , Spooncer E. , Parkinson E. K. , Lord B. I. , Allen T. D. , Dexter T. M.
Published: February 2005

When highly enriched multipotential spleen colony forming cells (CFU‐S) obtained following fluorescence activated cell sorting (FACS‐CFU‐S) are cultured on marrow stromal cells, they undergo proliferation and development to produce mature haemopoietic cells (Spooncer et al., Nature, 316:62–64, 1985). We now show that FACS‐CFU‐S behave in a similar way when cultured on monolayers of 3T3 cells, indicating that the 3T3 cells can supply at least part of the environment which is representative of marrow stromal cells and provide, therefore, a system for studying stromal cell: haemopoietic cell interactions. We also demonstrate that IL‐3‐dependent multipotential stem cell lines (FDCP‐Mix), but not a variety of other “committed” IL‐3‐dependent cell lines, resemble FACS‐CFU‐S in terms of their ability to proliferate and differentiate when cultured on 3T3 cells in the absence of IL‐3. In this system, attachment of the FDCP‐Mix to the 3T3 cells is critical for the subsequent maintenance of viability and stimulation of development of the cells. When the FDCP‐Mix cells are physically separated from the 3T3 cells, they die and their death cannot be prevented by using 3T3‐cell‐conditioned medium. The extracellular matrix generated by 3T3 cells is not sufficient for promoting attachment or viability of the FDCP‐Mix cells, indicating the importance of integral membrane components. However, attachment and development of FDCP‐Mix cells occurs on 3T3 cells that have been lightly fixed with glutaraldehyde indicating that active metabolism is not essential for the effects promoted by the 3T3 cells. We suggest that the ability of FACS‐CFU‐S and FDCP‐Mix cells to respond to 3T3 cells involves specific ligand/receptor interactions.
Interactions of dimethyl sulfoxide and granulocyte‐macrophage colony‐stimulating factors on the growth and maturation of HL‐60 cells
Authors: Brennan J. K. , Lee K. S. , Abboud C. N. , Erickson‐Miller C. L. , Keng P. C.
Published: February 2005

We have studied the interactions of dimethyl sulfoxide (DMSO), Giant Cell Tumor (GCT) cell‐conditioned medium (GCT CM), and highly purified granulocyte‐macrophage colony‐stimulating factors (GM‐CSF) on the growth and maturation of a highly passaged population of HL‐60 cells. DMSO produced dose‐dependent inhibition of HL‐60 growth in liquid and semisolid media. Growth was partially to completely restored by the addition of GCT CM to cultures. Experiments in which cell volume, cell cycle kinetics, tritiated thymidine (3HTdr) incorporation, cell number, and nitroblue tetrazolium (NBT) reduction were compared during culture indicated that DMSO inhibited the spontaneous increase in cell volume and flow of cells through the cell cycle which occurred in the first day of culture, the increase in 3HTdr incorporation which was detectable by day 2; and the increment in cell counts which occurred by day 3. These effects were opposed by GCT CM. In contrast, the DMSO‐induced increase in NBT reduction which occurred by day 6 was not influenced by GCT CM. The major principle opposing DMSO was GM‐CSF, since (1) highly purified GM‐CSF from GCT cells and recombinant GM‐CSF from COS cells transfected with the Mo cell GM‐CSF gene overcame greater than 50% of DMSO inhibition; and (2) conditioned media from cells not producing CSF, G‐CSF from GCT cells, and recombinant G‐CSF from Escherichia coli transfected with the G‐CSF gene from 5,637 cells were inactive. DMSO had little or no effect on the elaboration of autostimulatory activity by HL‐60 cells. DMSO is a useful agent for inhibiting the spontaneous growth of HL‐60 cells and restoring their dependence on GM‐CSF, a property which may be mediated through the effects of DMSO on cell cycle kinetics and/or maturation.
Quantitative ultrastructural, autoradiographic evidence for the magnitude and early involvement of the Golgi apparatus complex in the endocytosis of wheat germ agglutinin by cultured neuroblastoma
Authors: Mezitis Spyros G. E. , Stieber Anna , Gonatas Nicholas K.
Published: February 2005

Several ligands undergo endocytosis into the Golgi apparatus. We have examined with a quantitative ultrastructural, autoradiographic method the sequential endocytosis of tritiated wheat germ agglutinin (3H‐WGA) by cultured murine neuroblastoma cells. Cells were incubated with 3H‐WGA for 1 hour at 4°C, washed, and incubated in complete medium without ligand at 37°C for 5, 15, 30, and 120 minutes. At 5 minutes, the optimized sources/μm2 of neuroblastoma cell area, which represented the grain density of each compartment, were as follows: smooth vesicles and tubules, 1.03 ± 0.88; Golgi‐associated vesicles, i.e., clusters of vesicles within a 1 μm radius of the Golgi cisterns, 1.03 ± 0.31; Golgi cisterns, < 0.01; and lysosomes, 0.26 ± 0.16. At 15 minutes grain densities were: smooth vesicles and tubules, 0.9 ± 0.34; Golgi‐associated vesicles, 1.41 ± 0.28; Golgi cisterns, 0.73 ± 0.41; and lysosomes, 0.1 ± 0.09. At 30 minutes grain densities were: smooth vesicles and tubules, 0.46 ± 0.46; Golgi‐associated vesicles, 1.78 ± 0.34; Golgi cisterns, 0.89 ± 0.78; and lysosomes, 0.39 ± 0.14. At 2 hours, smooth vesicles, tubules, and Golgi cisterns were not labeled, Golgi‐associated vesicles were still labeled (0.71 ± 0.1), and lysosomes were heavily labeled (2.17 ± 0.22). These results are consistent with the hypotheses that either the Golgi complex (cisterns and associated vesicles) is an early and intermediate step of the endocytosis of 3H‐WGA into lysosomes or that it constitutes part of a separate and quantitatively significant pathway of endocytosis of this ligand.
Pathways of glutamine and glutamate metabolism in resting and proliferating rat thymocytes: Comparison between free and peptide‐bound glutamine
Authors: Brand Karl , von Hintzenstern Jutta , Langer Klaus , Fekl Werner
Published: February 2005

Pathways of glutamine metabolism in resting and proliferating rat thymocytes were evaluated by in vitro incubations of freshly prepared or 60‐h cultured cells for 1–2 h with [U14C]glutamine. Complete recovery of glutamine carbons utilized in products allowed quantification of the pathways of glutamine metabolism under the experimental conditions. Partial oxidation of glutamine via 2‐oxoglutarate in a truncated citric acid cycle to CO2 and oxaloacetate, which then was converted to aspartate, accounted for 76 and 69%, respectively, of the glutamine metabolized beyond the stage of glutamate by resting and proliferating thymocytes. Complete oxidation to CO2 in the citric acid cycle via 2‐oxoglutarate dehydrogenase and isocitrate dehydrogenase accounted for 25 and 7%, respectively. In proliferating cells a substantial amount of glutamine carbons was also recovered in pyruvate, alanine, and especially lactate. The main route of glutamine and glutamate entrance into the citric acid cycle via 2‐oxoglutarate in both cells is transamination by aspartate aminotransferase rather than oxidative deamination by glutamate dehydrogenase. In the presence of glucose as second substrate, glutamine utilization and aspartate formation markedly decreased, but complete oxidation of glutamine carbons to CO2 increased to 37 and 23%, respectively, in resting and proliferating cells. The dipeptide, glycyl‐L‐glutamine, which is more stable than free glutamine, can substitute for glutamine in thymocyte cultures at higher concentrations.
Effect of the mi allele on mast cells, basophils, natural killer cells, and osteoclasts in C57BI/6J mice
Authors: Stechschulte Daniel J. , Sharma Ram , Dileepan Kottarappat N. , Simpson Karen M. , Aggarwal Neelam , Clancy John , Jilka Robert L.
Published: February 2005

The osteopetrotic, microphthalmic (mi/mi) mouse lacks functional osteoclasts and has also been reported to be deficient in mast cells and natural‐killer (NK) cells. The later deficiencies could be secondary to the osteopetrotic marrow, or a direct result of the mi allele. Therefore, heterozygotes were examined for these cell types, since these mice do not exhibit osteopetrosis. Adult +/mi animals have approximately 50%, and mi/mi animals examined by histologic techniques or tissue histamine levels have 0–10%, of the peritoneal, dermal, and intestinal mast cells compared with that of +/+ animals. Leukocyte histamine, indicative of the number of basophils, demonstrates the same pattern. Histamine content per mast cell in +/+ and +/mi animals is identical. The number of large granular lymphocytes (LGL) in splenic leukocyte preparations from +/mi animals is 50% that of +/+ animals, and these cells are undetectable in preparations from mi/mi mice. NK activity against YAC‐1 cells paralleled the number of LGL present. The resorptive response of neonatal calvaria to parathyroid hormone was delayed in the case of cultured +/mi bone compared with that of +/+ bone, but the final rate of calcium release was identical. These data indicate that (1) the presence of one mi allele can affect the development of four distinct cell types, and (2) osteopetrosis alone does not account for the lack of mast cells, basophils, and NK cells in mi/mi mice.
Local effects of EGF, α‐TGF, and EGF‐like growth factors on lobuloalveolar development of the mouse mammary gland in vivo
Authors: Vonderhaar Barbara K.
Published: February 2005

Five‐week‐old female mice supplemented with estradiol and progesterone are able to respond to epidermal growth factor (EGF) and EGF‐like growth factors (α‐transforming growth factor [α‐TGF] and crude mammary‐derived growth factor) with local lobuloalveolar development when these growth factors are directly introduced into the mammary glands via slow‐release cholesterol‐based pellets. Contralateral glands receiving pellets containing only cholesterol showed no growth response. The local growth effect is maximal at 4–5 days of exposure to hormones and growth factors. The glands appear to be more sensitive to α‐TGF than EGF, since local development is seen with one‐fifth the level of the former vs. the latter growth factor and can be seen even in the absence of the systemic estrogen/progesterone supplement.
Reconstituted basement‐membrane matrix modulates fibroblast activities in vitro
Authors: Emonard H. , Grimaud J. A. , Nusgens B. , Lapière Ch. M. , Foidart J. M.
Published: February 2005

Cellular growth and collagen biosynthesis were compared in dermal calf fibroblasts cultured on plastic or on a reconstituted basement membrane gel, termed matrigel. This matrix, extracted from Engelbreth‐Holm‐Swarm tumors, consists mainly of laminin, entactin, type IV collagen, and heparan sulfate proteoglycan. The multiplication rate of fibroblasts grown on matrigel was stimulated compared to that of monolayered cells cultured on plastic, and these cells formed multilayers after 4 days. Protein and collagen biosynthesis was reduced in fibroblasts cultured on matrigel. A higher proportion of the newly synthesized collagen (40%) was incorporated to the extracellular matrix in cultures grown on matrigel than in those grown on plastic (14%). Type III collagen was the preferential collagen type deposited on matrigel, and the ratio type III:type I collagens secreted in the medium was also slightly higher in cultures grown on matrigel. Partially processed collagen was more abundant in fibroblasts grown on matrigel than in cells cultured on plastic. Finally, cells grown on matrigel exhibited a higher catabolic activity than cells grown on plastic. In this experimental model, the reconstituted basement‐membrane matrix seems to influence the activities of fibroblasts significantly.
Oxygen and substrate dependence of hepatic cellular respiration: Sinusoidal oxygen gradient and effects of ethanol in isolated perfused liver and hepatocytes
Authors: Kekonen Eija M. , Jauhonen V. Pekka , Hassinen Ilmo E.
Published: February 2005

The oxygen dependence of hepatic cellular respiration was studied by employing simultaneous organ spectrophotometry of cytochromes and hemoglobin, the latter used as an intrasinusoidal optical oxygen probe. The Kmof cytochromeaa3for oxygen was found to be 6.8 μM in the isolated perfused liver and 0.3 μM in suspensions of isolated hepatocytes. The results indicate that the sinusolid‐to‐cell pO2gradient is about 5 torr. Optical determination of the average effective pO2indicates that the axial sinusoidal O2profile does not conform to zero‐order O2uptake in the liver. Because of extensive NAD+reduction, ethanol increases the thermodynamic driving force of oxidative phosphorylation, and it also increased the oxygen consumption in both the perfused liver and the hepatocyte suspension, but had no effect on the grade of steady‐state cytochromeaa3reduction, the cellular energy state [ATP]/[ADP]·[Pi], or the Kmof cytochromeaa3for oxygen. The results indicate that hepatic energy metabolism is oxygen independent at very low O2concentrations, but that the sinusoidal axial O2concentration is anomalous, probably due to the spatial arrangement of the metabolizing systems.
Effect of sodium butyrate on S‐100 protein levels and the cAMP response
Authors: Hirschfeld Alan , Bressler Joseph
Published: February 2005

Sodium butyrate (NaB), when added to cell cultures, produces a variety of morphological and biochemical changes. We examined its effects, in nM concentrations, on the expression to two glioma cell‐associated proteins, glial fibrillary acidic protein (GFAP) and S‐100 protein in human glioma‐derived cell line (RF), and of S‐100 protein in the C6 rat glioma cell line. GFAP levels decreased by about 50% in the RF cell line, and S‐100 protein levels decreased protein levels decreased by about 40% after treatment with 1 mM NaB for 48 h. In the C6 rat glioma cell line, isoproterenol with theophylline was found to increase S‐100 protein levels by two‐fold over basal levels. NaB was found to inhibit the induction of S‐100 protein but exhibited no effect no effect on the basal levels of the protein. Other short chain fatty acids, including sodium propionate and sodium isobutryate, exhibited partial inhibitory activity. NaB, at an EC50 of 1 mM, was also found to inhibit both the beta‐adrenergic and the forskolin‐mediated increase in cAMP levels in these cells. This suggests that NaB may inhibit cells from expressing S‐100 protein by attenuating cAMP levels.
Mechanism of sucrose uptake by isolated rat hepatocytes
Authors: Sasaki Anna W. , Williams Stuart K. , Jain Mahendra , Wagner Roger C.
Published: February 2005

The transport of molecules by nonspecific endocytosis has been described in many cell types, but it has not been characterized in hepatocytes. Because of its central role in the clearance of solutes from portal blood, endocytosis might represent a significant mode of cellular transport. We investigated the mechanism of sucrose uptake in an isolated hepatocyte system. Liver cells were isolated by perfusion and collagenization of rat liver, followed by differential centrifugation. Hepatocytes were then incubated with 14C‐sucrose and harvested by spinning through oil in microfuge tubes. Radioactivity was standardized against DNA content. We found that sucrose uptake is concentration‐dependent from 5 μM to 100 mM and follows first‐order kinetics. Washout studies indicate that exocytosis is responsible for the dynamic equilibrium reached. Arrhenius analysis of temperature dependence yields a linear plot (Ea = 14.2 Kcal/mol). In addition, sucrose uptake is independent of cellular ATP levels. We conclude that sucrose is transported by fluid‐phase micropinocytosis in isolated hepatocytes and that this transport mechanism may be important in the uptake of diverse molecules into liver cells.
Intraclonal diversity of fibrosarcoma cells for the production of macrophage colony‐stimulating factor and granulocyte colony‐stimulating factor
Authors: Sakai Nobuo , Kubota Masao , Shikita Mikio , Yokota Masahiko , Ando Koichi
Published: February 2005

A new cell line was established from fibrosarcoma that had spontaneously developed in a mouse. The cells were maintained growing in cultue for two years and constantly produced both macrophage colony‐stimulating factor (M‐CSF) and granulocyte colony‐stimulating factor (G‐CSF). Cloning of the cells by anchorage‐independent colony formation gave subclones showing the activity of producing M‐CSF and G‐CSF in different proportions, whereas no subclone produced G‐CSF without producing M‐CSF simultaneously. Recloning of the bipotential subclones again gave clonal derivatives producing two types of CSF in various proportions. The observed heterogeneity of the cloned cells seems to be an epigenetic phenomenon, because the cells resumed the G‐CSF producing activity in the absence of cell proliferation. After equilibrium was achieved, all of the subclones produced both M‐CSF and G‐CSF nearly in equal proportions. Tumorigenic and leukocytosis‐inducing activity of the cloned cells was nearly comparable with the activity of the original tumor cells.
In vitro effect of a thymic epithelial culture supernate or thymosin fraction 5 on rabbit platelet aggregation and intracellular cyclic AMP levels
Authors: Hashimoto Shuichi , Itoh Yukie , Kawaguchi Akiko , Mizuno Yoshio , Muraoka Matuo , Tsuchiya Masaharu
Published: February 2005

Supernates of thymic epithelial cell culture (STEC) strongly inhibit aggregation induced by addition of adenosine diphosphate (ADP: 1 μM) or thrombin (0.5 unit per ml) to washed platelet suspensions and accelerated the restoration from ADP‐triggered aggregation. At the same time, STEC increased the level of platelet adenosine 3′,5′‐cyclic monophosphate (cyclic AMP) in a dose‐dependent manner. Depending on the concentration used, thymosin fraction 5 increased the level of intracellular cyclic AMP ranging between 5 and 100 μg per ml, as well as inhibiting ADP‐induced platelet aggregation. The activities of both STEC and thymosin fraction 5 were found to act exclusively on cyclic AMP phosphodiesterase activity in platelets. In contrast the supernates from Chang, HeLa, or HCC‐M cells did not affect platelet aggregation induced by ADP, but slightly increased the cyclic AMP level (Chang, HeLa). Within 2 min after the treatment with STEC, more than 50% of the maximum inhibitory activity on platelet aggregation and increases in intracellular cyclic AMP were observed. These activities disappeared following STEC treatment with pronase E. STEC activity was found predominantly in the 1,000–50,000‐dalton fractions. These activities were not altered when STEC was treated by adenosine deaminase. The level of prostaglandin E (PGE) derivaties in STEC was about two times that found in the control culture medium. These data suggest that the biological activity of STEC in the platelets might be attributed to thymosinlike polypeptides and PGE1.