bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment and characterization of a unique human cell line that proliferates dependently on GM‐CSF, IL‐3, or erythropoietin
Authors: Kitamura Toshio , Tange Tsuyoshi , Terasawa Takashi , Chiba Shigeru , Kuwaki Tomoaki , Miyagawa Kiyoshi , Piao Yun‐Feng , Miyazono Kohei , Urabe Akio , Takaku Fumimaro
Published: February 2005

We have established a novel cell line, designated as TF‐1, from a patient with erythroleukemia, which showed complete growth dependency on granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or on interleukin‐3 (IL‐3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short‐term growth of TF‐1, but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF‐1 synergistically. Transforming growth factorβ and interferons inhibited the factor‐dependent growth of TF‐1 cells in a dose‐dependent fashion, and monocyte‐colony stimulating factor and interkeukin‐1 enhanced the GM‐CSF‐dependent growth of TF‐1. Ultrastructural studies revealed some very immature features in this cell line. Although TF‐1 cells do not express glycophorin A or carbonyl anhydrase I, the morphological and cytochemical features, and the constitutive expression of globin genes, indicate the commitment of TF‐1 to erythroid lineage. When induced to differentiate, TF‐1 entered two different pathways. Specifically, hemin and delta‐arninolevulinic acid induced hemoglobin synthesis, whereas TPA induced dramatic differentiation of TF‐1 into macrophage‐like cells. In summary, TF‐1 is a cell lineof immature erythroid origin that requires GM‐CSF, IL‐3, or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage‐like cells. TF‐1 is auseful tool for analyzing the human receptors for IL‐3, GM‐CSF, and EPO or the signal transduction of these hemopoietic growth factors.
Nutritional folate deficiency in chinese hamster ovary cells I. Characterization of the pleiotropic response and its modulation by nucleic acid precursors
Authors: Borman Linda S. , Branda Richard F.
Published: February 2005

Nutritional folate deficiency in Chinese hamster ovary (CHO)‐K1 cells inhibited population growth rate and caused growth arrest within 3 days of culture in Fol‐medium [without folate, hypoxanthine (Hx), and thymidine (TdR)]. Coincident with impaired population growth was a transient delay in cell cycle progression through S phase and an increase in cell size. The growth‐arrested population of predominantly G1 phase cells exhibited an increased adhesion to the culture substratum. There was a time‐dependent loss of cell reproductive capacity. All these various perturbations of cellular phenotype induced by folate deficiency were prevented by the addition of folate or a combination of TdR and Hx to the Fol‐medium. However, the singular presence of each nucleotide precursor differentially affected the pleiotropic response. The addition of Hx to Fol‐medium exacerbated the aforementioned abnormalities, producing a threefold increase in mean cell volume, a 72 hr accumulation of cells in the S phase of the cell cycle, and a rapid demise in cell clonogenicity. Unexpectedly, we found reduced cell adhesion in these cultures. In contrast, folate‐deficient cells supplemented with TdR exhibited a general amelioration of cell perturbations with respect to cell size, cell cycle distribution, and reproductive viability. Notably, such populations were not released from growth inhibition or subsequent growth arrest, and the cells became elongated and highly adherent with time. When cell populations from each of the three conditions of folate‐deficient culture were released from growth arrest by addition of complete medium, the respective profiles of synchronous cell cycle progression were distinctive.
Statin, a nonproliferation‐specific protein, is associated with the nuclear envelope and is heterogeneously distributed in cells leaving quiescent state
Authors: Wang Eugenia
Published: February 2005

Statin, a protein of 57,000 daltons, is present primarily in the nuclei of nonproliferating cells of terminally differentiated tissues or of in vitro aged fibroblast cultures. In young growing cells, the protein can be induced to appear in the nuclei once the cell‐cycle traverse is blocked by various tissue culture manipulations, such as serum starvation; this expression, however, can be rapidly removed by addition of serum. The disappearance of statin in cells leaving the quiescent state is not uniform along the periphery of the nucleus; it can be distributed in various patterns, such as caps, nodules, patches, or irregular granules. This unusual distribution seems to suggest that preferential sites exist at the region of the nuclear envelope where statin presence may residually remain. The concentration of statin at the nuclear envelope region in cells at GO‐quiescent phase is confirmed by the intense staining of fluorescent antibody at the periphery of isolated rat liver nuclei. Further examination of the isolated nuclei reveals that the protein is associated with the lamina compartment of the nuclear envelope; this is evidenced by the results of immunoblotting experiments showing statin presence in the fraction enriched for lamins A‐C. Immunogold labelling studies show that the protein is located in the general area of the nuclear envelope. These results suggest that statin in GO‐quiescent cells is located predominantly at the nuclear envelope region and that in this vicinity there may exist geometrically sites of statin concentration as evidenced by the heterogeneous distribution in those cells experiencing the departure from the quiescent state.
Stabilization by heparin of acidic fibroblast growth factor mitogenicity for human endothelial cells in vitro
Authors: Mueller Stephen N. , Thomas Kenneth A. , Salvo Jerry Di , Levine Elliot M.
Published: February 2005

The effects of heparin and other glycosaminoglycans (GAGs) on the mitogenicity and stability of acidic fibroblast growth factor (aFGF) were studied. The mitogenic activity of aFGF was assayed utilizing cultured adult human endothelial cells (AHECs) isolated from iliac arteries and veins as target cells. In most experiments, aFGF purified from bovine brain was employed; in some experiments recombinant bovine aFGF was used and qualitatively similar results were obtained. In the presence of heparin, bovine aFGF at doses between 0.5 and 1.0 ng/ml (30‐60 pM) elicited half the maximum AHEC growth over a 4‐day period depending on the cell line tested; in the absence of heparin, significant growth was not observed at aFGF concentrations less than 10‐20 ng/ml. This effect of heparin was dose‐dependent over the range 0.1‐10 μg/ml (half‐maximum dose, 2 μg/ml). The mitogenic activity of bovine aFGF for AHECs decreased by 50% after preincubation in culture medium without cells at 37°C for 2 ½ to 3 hours. In contrast, the mitogenic activity of bovine aFGF preincubated in the presence of heparin‐containing culture medium without cells was dramatically stabilized (half‐life 24‐29 hours). These effects also were observed in serum‐free medium. Several GAGs structurally related to heparin such as chondroitin‐4‐sulfate, chondroitin‐6‐sulfate, dermatan sulfate, and hyaluronic acid neither potentiated nor stabilized aFGF mitogenic activity. However, heparan sulfate from bovine lung was found to be nearly as active as heparin in both these effects. These data suggest that the binding and stabilization of mitogens by extracellular and tissue‐associated heparan sulfates might play important roles in the regulation of AHEC growth.
HSP70‐related proteins in bovine skeletal muscle
Authors: Guerriero Vince , Raynes Deborah A. , Gutierrez Jesus A.
Published: February 2005

Constitutive expression of HSP70‐related proteins was detected in a variety of bovine tissues using a specific antibody. All tissues contained a 73 kilodalton protein. A lower molecular weight form (72 kilodaltons) that co‐migrated on two‐dimensional gels with the stressed‐induced HSP70 was present in high levels in bovine skeletal muscle, but absent from rat skeletal muscle. Two‐dimensional gel analysis revealed several isoforms for both the 73 and 72 kilodalton forms. Purification of HSP70‐related proteins from bovine skeletal muscle, thymus gland and rat skeletal muscle demonstrated that the antibody recognized all the forms present in the tissue homogenates. The two proteins are similar but distinct as detected by one‐dimensional peptide mapping. The lower molecular form was not present in fetal tissue but was detectable in newborn animals, suggesting that the levels are regulated during development.
Elastin repeat peptides as chemoattractants for bovine aortic endothelial cells
Authors: Long Marianna M. , King Vickie J. , Prasad Kari U. , Freeman Bruce A. , Urry Dan W.
Published: February 2005

Cultured bovine aortic endothelial cells migrate toward a concentration gradient of repeating elastin peptides, specifically the repeating nonamers Gly‐Phe‐Gly‐Val‐Gly‐Ala‐Gly‐Val‐Pro and Gly‐Leu‐Gly‐Val‐Gly‐Ala‐Gly‐Val‐Pro and the repeating hexamer Val‐Gly‐Val‐Ala‐Pro‐Gly. Dose‐response experiments demonstrate that the peak of activity occurs at 8 × 10‐8 M for the nonapeptides and 1 × 10‐8 M for the hexapeptide. Checkerboard assays establish that the movement is chemotaxis and not chemokinesis. Because of the concentration difference in the responsiveness between the nonapeptide and the hexapeptide, the cells can differentiate between the two types of repeats. The positive control for the chemo‐taxis studies was fibronectin.
Sequential expression of cell surface C3bi receptors during neutrophil locomotion
Authors: Francis Joseph W. , Todde Robert F. , Boxer Laurence A. , Petty Howard R.
Published: February 2005

Fluorescence microscopy has been used to study the cell surface distribution of the complement receptor for C3bi (CR3) on human neutrophils during locomotion. CR3 is an integral membrane protein that participates in cell attachment phenomena including chemotaxis. Fluorescein‐ and rhodamine‐conjugated monoclonal IgG or Fab fragments were used to label CR3. We have previously shown that CR3 is uniformly distributed on unstimulated cells. During cell locomotion the fluorescent labels redistribute to the uropod and retraction fibers. To better understand the role of CR3 in chemotaxis, we have performed sequential two‐color labeling experiments in conjunction with fluorescence microscopy. Double‐labeling experiments were conducted by labeling adherent neutrophils with fluorescein‐conjugated anti‐CR3 followed by chemotaxis in a gradient of FMLP (10‐7 M). The cells were then labeled again with rhodamine‐conjugated anti‐CR3. The uropod and distal training filopodia were labeled with fluorescein, whereas the cell body and occasionally proximal filopodia near the uropod were labeled with rhodamine. When neutrophils were fixed and permeabilized prior to the second CR3 labeling, the second fluorescent label was localized to a granule‐like compartment(s), often near the lamellipodium. The results suggest a flow of CR3 from intracellular granules → lamellipodia and cell body → uropod → trailing filopodia during chemotaxis.
Effects of platelet‐derived growth factor on bone formation in vitro
Authors: Canalis Ernesto , McCarthy Thomas L. , Centrella Michael
Published: February 2005

Platelet‐derived growth factor (PDGF) is a polypeptide found in a variety of tissues, including bone, where it could act as an autologous regulator of skeletal remodeling. Therefore, a recombinant B chain homodimer of human PDGF was studied for its effects on bone formation in cultured rat calvariae. PDGF at 10‐100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to sixfold and increased the DNA content and the number of colcemid‐induced metaphase arrested cells. This effect was observed in the fibroblast and precursor cell‐rich periosteum. As a result of its mitogenic actions, PDGF enhanced [3H]proline incorporation into collagen, an effect that was observed primarily in the osteoblast‐rich central bone. The effect of PDGF was not specific for collagen since it also increased noncollagen protein synthesis. In addition, PDGF increased bone collagen degradation. PDGF and insulin‐like growth factor (IGF) I had additive effects on calvarial DNA synthesis, but PDGF opposed the stimulatory effect of IGF I on collagen synthesis and IGF I prevented the PDGF effect on collagen degradation.

In conclusion, PDGF stimulates calvarial DNA synthesis which causes an increased number of collagen‐synthesizing cells, but PDGF also enhances bone collagen degradation.
Heparin‐derived oligosaccharides: Affinity for acidic fibroblast growth factor and effect on its growth‐promoting activity for human endothelial cells
Authors: Bârzu Tereza , Lormeau Jean‐Claude , Petitou Maurice , Michelson Susan , Choay Jean
Published: February 2005

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra‐ to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose‐immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a “regular” hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa‐ and decasaccharides were also retained by the column. The Oligosaccharides eluted by 1 M NaCI from the affinity column (“high‐affinity” Oligosaccharides) and those washed from the column at 0.2 M NaCI (“low‐affinity” Oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all Oligosaccharides tested, except the “regular” disaccharide, protected aFGF againsttrypsin and collagenase digestion. At higher ionic strength (> 0.2 M NaCI), only high‐affinity Oligosaccharides showed a protective effect. The high‐affinity oligosaccharides (hexa‐ to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1‐5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF‐like activity.
Adenosine triphosphate, bradykinin, and thyrotropin‐releasing hormone regulate the intracellular Ca2+ concentration and the 45Ca2+ efflux of human thyrocytes in primary culture
Authors: Raspè E. , Andry G. , Dumont J. E.
Published: February 2005

The hormonal stimulation of phospholipase C and the consequent activation of the Ca2+‐phosphatidylinositol cascade in eukaryotic cells is associated with modifications of the [Ca2+] (intracellular Ca2+ concentration) which modulates cellular functions. In this study, these modifications were investigated in primary cultures of human thyroid cells.

The mean apparent basal [Ca2+] of human thyrocytes measured using the intracellularly trapped fluorescent indicator Quin‐2 was found to be 89°C 16 nM (n = 49). ATP and, to a lesser extent, ADP, but not AMP or adenosine, elicited a concentration‐dependent biphasic rise in human thyrocytes [Ca2+] and increased their 45Ca2+ efflux. The first transient phase of the [Ca2+] rise induced by ATP was resistant to extracellular Ca2+ depletion, whereas the second sustained phase was abolished in these conditions. This suggests that although the first phase of this response involves a release of Ca2+ from intracellular stores, the second phase requires extracellular Ca2+ influx. The response of human thyro‐cytes to analogs of ATP is compatible with a P2‐purinergic effect of ATP on these cells. Bradykinin and TRH affected the human thyrocyte [Ca2+] and 45Ca2+ efflux similarly to ATP. The human thyrocyte [Ca2+] and the 45 Ca2+ efflux were not modified by carbachol, a nonhydrolyzable analog of acetylcholine.

The present results suggest the presence of P2‐purinergic receptors to ATP and of receptors to TRH and bradykinin on human follicular thyroid cells. They also confirm that the Ca2+‐phosphatidylinositol cascade is present in these cells and suggest that this cascade is modulated by ATP, TRH, and bradykinin. As this cascade is involved in the regulation of protein iodination, and therefore of thyroid hormones synthesis, these agents might have an important role in the regulation of the thyroid function.
Neutral amino acid transport in human synovial cells: Substrate specificity of adaptative regulation and transinhibition
Authors: Aussel Christian , Rousseau‐Loric Sophie , Cynober Luc , Agneray Jean , Ekindjian Ohvanesse G.
Published: February 2005

Neutral amino acid transport was characterized in human synovial cells. The amino acids tested are transported by all three major neutral amino acid transport systems, that is, A, L, and ASC. The model amino acid 2‐aminoisobutyric acid (AIB) was found to be a strong specific substrate for system A in synovial cells. When cells were starved of amino acids, the activity of AIB transport increased, reaching a maximum within 1 h. The stimulation of transport activity was not blocked by cycloheximide and would thus appear to be related to a release from transinhibition. Similarly, the decrease in the activity of AIB transport observed after the addition of α‐methyl‐aminoisobutyric acid (meAIB) appeared to be related to transinhibition. However, using a different approach, that is, amino acid starvation followed by incubation with 10 mM meAIB and transfer to an amino acid‐free medium with or without cycloheximide supplementation, a clear increase in AIB uptake, due both to derepression and a release from transinhibition, was observed. Unlike human fibroblasts, the derepression of system A in these synovial cells was not serum‐dependent. The process of derepression was observed only after preloading with meAIB. Neither AIB nor alanine produced this phenomenon. Moreover, alanine preloading led to a large increase in AIB transport activity due to a release from transinhibition. These observations indicate that the process of derepression and release from transinhibition are specific to the substrates present in the culture medium prior to amino acid starvation.
Induction of polarized apical expression and vectorial release of carcinoembryonic antigen (CEA) during the process of differentiation of HT29‐D4 cells
Authors: Fantini Jacques , Rognoni Jean‐Baptiste , Culouscou Jean‐Michel , Pommier Gilbert , Marvaldi Jacques , Tirard Alain
Published: February 2005

HT29‐D4 clonal cells can be induced to differentiate by a simple alteration of the culture medium, that is, by the replacement of glucose by galactose [Fantini, J., et al. (1986) J. Cell Sci., 83:235–249] as reported for the nonclonal HT29 cells [Pinto, M., (1982) Biol. Cell, 44:193–196]. An essential property of the HT29‐D4 cell line is the fact that no cell loss occurs after the medium change, so that the differentiated cells can be considered as the true counterpart of the undifferentiated one. This model is particularly suitable to study morphological and biochemical events associated with the progressive establishment of the differentiation state.

We report here that Carcinoembryonic antigen (CEA), a 180 kDa glycoprotein originally described as a colon tumor associated antigen, is faintly expressed at the surface of undifferentiated HT29‐D4 cells. These cells release a small amount of CEA (2.5 ng/106 cells/24 hr) in the culture medium. Fourty‐eight hours after glucose substitution by galactose, both CEA cell surface expression and release are strongly enhanced as demonstrated by immunofluorescence and immuno‐precipitation studies. Ten days after the medium change, the amount of CEA released reaches a maximum value of 130 ng/106 cells/24 hr, which remains stable for differentiated HT29‐D4 cells cultured in glucose‐free, galactose‐containing medium (Gal‐medium) for several months.

HT29‐D4 cells grown in Gal‐medium in porous‐bottom culture dishes generate leakproof epithelial monolayers. We have successfully performed an independent radioiodination of the apical and basolateral domains of these cells, followed by immunoprecipitation. We demonstrate that CEA is expressed exclusively at the apical surface of differentiated HT29‐D4 cells, since the 180 kDa polypeptide was immunoprecipitated only when the radioiodination was performed at the apical side of the monolayer.

Leakproof HT29‐D4 monolayers cultured in permeable chambers were also used to demonstrate that CEA was exclusively released in the medium bathing the apical side of the cells. In conclusion, this study of cell surface CEA expression and CEA release during the process of differentiation of HT29‐D4 cells demonstrated that (1) CEA cell surface expression and CEA release are correlated with cell differentiation; (2) CEA is expressed in the apical brush border membrane of differentiated HT29‐D4 cells; and (3) CEA release is exclusively oriented toward the apical side of the polarized monolayer.
Possible role of prostaglandins in the regulation of mouse myoblasts
Authors: Rossi Michael J. , Clark Mike A. , Steiner Sheldon M.
Published: February 2005

A differentiation‐defective mouse myoblast subclone (DD‐1), cells of which do not fuse into myotubes nor synthesize muslce‐specific proteins, was employed to help define the role of eicosanoids in mouse myoblast differentiation. We observed by hplc, tIc, and radioimmunoassay that the DD‐1 cells release strikingly higher levels of cyclooxygenase pathway products prostaglandin E2 and F2α into the culture medium than the parental non‐differentiation‐defective cells (DZ). In contrast, the levels of 15‐hydroxyeicosatetraenoic acid (15‐HETE), a lipoxygenase product, and a putatively identified second lipoxygenase product (LLP) did not differ greatly in the two cell types. The DD‐1 cells also have strikingly higher levels of cyclooxygenase activity than the parental cells as determined by intact and broken cell assays. Additional fusion‐defective clones were isolated on the basis of their flattened appearance and ability to grow in “mitogen‐poor” medium and these cells also released strikingly higher levels of prostaglandins E2 and F2α into the growth medium. The “turn on” of the cyclooxygenase pathway in the DD‐1 cells and other fusion‐defective cells is consistent with the hypothesis that the products of this pathway contribute to the inability of myoblasts to fuse with one another. This hypothesis is supported by the observation that there is a dose‐dependent decrease in fusion of DZ cells when PGE2 is added to commitment medium.
Intranuclear distribution of the human myeloid cell nuclear differentiation antigen in HL‐60 cells
Authors: Duhl David M. , Gaczynski Marek , Olinski Ryszard , Briggs Robert C.
Published: February 2005

Based on solubility properties, the human myeloid cell nuclear differentiation antigen exists as at least two distinct populations. Most is easily extracted from isolated nuclei in 0.35 M NaCl, while 20 percent resists such treatment. Compared to undigested nuclei, both the amount of myeloid cell nuclear differentiation antigen (MNDA) released from nuclei after DNase I treatment and the amount resisting further extraction in 0.35 M NaCl increased after DNA was digested with DNase I. Under these conditions, there was a concomitant decrease in the amount of MNDA that was extractable with 0.35 M NaCl. Mixing nuclear protein extracts that contain MNDA with nuclei from cells that do not express this protein demonstrated that the MNDA redistributes from the freely soluble form to the nuclear residual fraction as a consequence of DNase I digestion. These data are consistent with a model in which the amount of MNDA that is tightly bound to salt‐washed nuclei is held constant in the presence of an excess of unassociated MNDA in the nucleus, and that the level of MNDA binding to this nuclear fraction increases in proportion to the extent of DNA damage resulting from DNase I digestion.
Interleukin‐1 is a potent regulator of JE and KC gene expression in quiescent BALB/c fibroblasts
Authors: Hall David J. , Brownlee Clare , Stiles Charles D.
Published: February 2005

Interleukin‐1 alpha and beta are polypeptide hormones with a broad range of biological activities. Both interleukins are recognized by a receptor that has been characterized as a member of the immunoglobin superfamily. The interleukin‐1 receptor does not appear to be a tyrosine protein kinase. Moreover, the intracellular events that mediate the multiple interleukin‐1 responses are poorly understood. Here we show that the JE and KC genes, first isolated and characterized as platelet‐derived growth factor inducible in quiescent BALB/c‐3T3 fibroblasts, are induced by femtomolar concentrations of recombinant interleukin‐1 alpha (rlL‐1). The response of JE and KC to IL‐1 occurs at the transcriptional level. These observations suggest that an analysis of the JE and KC transcriptional response to rlL‐1 may aid in identifying elements involved in interleukin‐1‐mediated signal transduction.
Maintenance of proximal and distal cell functions in SV40‐transformed tubular cell lines derived from rabbit kidney cortex
Authors: Vandewalle A. , Lelongt B. , Geniteau‐Legendre M. , Baudouin B. , Antoine M. , Estrade S. , Chatelet F. , Verroust P. , Cassingena R. , Ronco P.
Published: February 2005

This paper reports the preparation and describes the properties of three renal tubular cell lines derived using SV40 infection of primary cultures of rabbit kidney cortical cells, enriched in proximal cells. RC.SV1 was initially derived from cultures grown in the presence of fetal calf serum exhibiting a low degree of proximal differentiation. The cells were subsequently adapted to grow in serum‐free hor‐monally defined medium and display basic properties of proximal tubule cells including well‐developed apical microvilli, strong expression of brush‐border hydrolases, Na+‐coupled glucose uptake, and increased cyclic AMP production when exposed to PTH. The other two cell lines were derived from cultures in serum‐free hormonally defined medium and propagated in the same medium. They are characterized by some common properties including rare and short microvilli, low expression of apical hydrolases, and low or undetectable Na+‐dependent glucose uptake, but differ by their abilities to respond by an increase in cAMP to various hormonal stimuli. RC.SV2 cells are sensitive to calcitonin and to a lesser extent to isoproterenol and PTH, suggesting that they may originate from the thick ascending limb of Henle's loop and the bright portion of the distal tubule. RC.SV3 responds essentially to isoproterenol and arginine va‐sopressin, suggesting a more distal origin (late distal and initial collecting tubule). Emergence of distal cell lines from cultures exhibiting proximal characteristics may be related to distal cell overgrowth as suggested by analysis of growth kinetics and increased Na+/H+ exchanger activity in RC.SV2 compared with RC.SV1.
Lipocortin I (p35) is abundant in a restricted number of differentiated cell types in adult organs
Authors: Fava Roy A. , McKanna James , Cohen Stanley
Published: February 2005

Lipocortin‐l (p35) is a unique calcium‐ and phospholipid‐binding protein of the lipocortin/calpactin family. Although several possibilities have been suggested, functions for the individual proteins of this family are not yet known with certainty. As an initial step in the identification of the biological function(s) of p35, we have used immunohistochemical methods to define precisely many of the cellular phenotypes that contain p35 in vivo. In all organs where p35 is found, we have observed a striking distribution of p35‐positive cells. Typically it is highly enriched in a limited range of differentiated cell types while apparently totally absent from most others. Our identification of specific p35‐positive cell types in vivo will now set limitations on likely possibilities for functions of this protein and thereby permit a more logical approach to the determination of its true function.
Transforming growth factors type β1 and β2 are equipotent growth inhibitors of human breast cancer cell lines
Authors: Zugmaier Gerhard , Ennis Bruce W. , Deschauer Bernd , Katz Deborah , Knabbe Cornelius , Wilding George , Daly Peter , Lippman Marc E. , Dickson Robert B.
Published: February 2005

At least one member of the TGF‐β family, TGF‐β1, has been previously shown to inhibit the anchorage‐independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF‐β family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF‐β1 and TGF‐β2 on the anchorage‐independent growth of the estrogen receptor‐negative cell lines MDA‐MB‐231, SK‐BR‐3, Hs578T, MDA‐MB‐468, and MDA‐MB‐468‐S4 (an MDA‐MB‐468 clone not growth inhibited by EGF) and the estrogen receptor‐positive cell lines MCF7, ZR‐75‐1, T‐47D. TGF‐β1 and TGF‐β2 caused a 75–90% growth inhibition of MDA‐MB‐231, SK‐BR‐3, Hs578T, and MDA‐MB‐468 cells and a 50% growth inhibition of ZR‐75‐1 and early passage (< 100) MCF7 cells. T‐47D cells responded to TGF‐β only in serum‐free conditions in the presence of IGF‐1 or EGF. The growth of MDA‐MB‐468‐S4 cells and late passage (> 500) MCF7 cells was not inhibited by TGF‐β1 or TGF‐β2. TGF‐β‐sensitive MCF7 and MDA‐MB‐231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF‐β‐related polypeptide. TGF‐β1 and TGF‐β2 were mutually competitive for receptor binding with a similar affinity (Kd 25‐130 pM, 1,000‐13,000 sites per cell). To determine the time course of the TGF‐β effect, an anchorage‐dependent growth assay was carried out using MDA‐MB‐231 cells. Growth inhibition occurred at 6 days, and cell‐cycle changes were seen 12 hr after the addition of TGF‐β. Cells accumulated in the G1 phase and were thus inhibited from entering the S‐phase. These data indicate that TGF‐β is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF‐β effects in vivo.
Identification of alpha transforming growth factor as a possible local trophic agent for the mammary gland
Authors: Smith J. A. , Barraclough R. , Fernig D. G. , Rudland P. S.
Published: February 2005

Biologically active alpha‐transforming growth factor (α‐TGF) has been identified in medium conditioned by rat mammary myoepithelial and, to a lesser extent, by epithelial cell lines in culture and in the rat mammary gland. The α‐TGF has been identified by its wide spectrum of activity in promoting growth of mammary‐derived cells in vitro, by its chromatographic behaviour on reversed‐phase high‐performance liquid chromatography (HPLC), by its competition with epidermal growth factor (EGF) for the EGF receptor, and by the presence of messenger RNA for α‐TGF in the secreting cells. In vivo the amount of α‐TGF isolated is sixfold greater from the mammary glands of lactating than from those of virgin female rats. It is proposed that α‐TGF is produced by the myoepithelial cells of the mammary gland, as a local trophic agent that stimulates growth of the various cell types of the gland.
Cyclic adenosine monophosphate acts synergistically with dexamethasone to inhibit the entrance of cultured adult rat hepatocytes into S‐phase: With a note on the use of nucleolar and extranucleolar [3H]‐thymidine labelling patterns to determine rapid changes in the rate of onset of DNA replication
Authors: Vintermyr Olav Karsten , Mellgren Gunnar , Bøe Roald , Døskeland Stein Ove
Published: February 2005

Analogs of cyclic adenosine monophosphate (cAMP) (N6benzoyl cAMP and N6monobutyryl cAMP) as well as agents that increased the intracellular level of cAMP (glucagon and isobutylmethylxanthine) inhibited the EGF‐stimulated DNA replication of adult rat hepatocytes in primary culture independently of cell density. This inhibition was strongly potentiated by the glucocorticoid dexamethasone. The effect of cAMP (and dexamethasone) was not due to toxicity, because the inhibition was reversible and the cell ultrastructure preserved. cAMP acted by decreasing the rate of transition from G1‐ to S‐phase, the duration of G2‐ and S‐phase of the hepatocyte cell cycle being unaffected. DNA replication started in the extranucleolar compartment of the nucleus and ended in the nucleolar compartment as described earlier for cells grown in the absence of cAMP (O.K. Vintermyr and S.O. Døskeland, J. Cell. Physiol., 1987, 132:12‐21). The action of cAMP was very rapid: significant inhibition of the transition was noted 2 hr after the addition of glucagon/IBMX and half‐maximal inhibition after 4 hours. The determination of extranucleolarly labelled nuclei in cells pulse‐labelled with [3H]thymidine allowed precise analysis of rapid changes in the probability of transition from G1‐ to S‐phase. The extranucleolar labelling index could also be determined in cells continuously exposed to [3H]thymidine.
Inhibition of epidermal growth factor/transforming growth factor‐α–stimulated cell growth by a synthetic peptide
Authors: Eppstein Deborah A. , Marsh Y. Vivienne , Schryver Brian B. , Bertics Paul J.
Published: February 2005

Estrogen‐stimulated growth of the human mammary adenocarcinoma cell line MCF‐7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high‐affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor‐α (TGF‐α)‐stimulated growth of these MCF‐7 cells. An analogous pattern of specific EGF or TGF‐α growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF‐α, but containing additional modifications designed for increased membrane affinity ([Ac‐D‐hArg(Et), Gly32,33]HuTGF‐α(31‐43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF‐α, or estrogen‐stimulated growth of MCF‐7 cells but did not inhibit insulin‐like growth factor‐1 (IGF‐1)‐stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen‐stimulated growth of these MCF‐7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF‐α‐like growth factors. The TGF‐α peptide likewise inhibited EGF‐ but not fibroblast growth factor (FGF)‐ or platelet‐derived growth factor (PDGF) ‐stimulated growth of NIH‐3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0‐arrested cells, nor did it effect growth of NR‐6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF‐stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF‐α peptide did, however, affect the structure of laminin as manifested by laminin self‐aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF‐mediated cell growth.
Altered responses of regenerating hepatocytes to norepinephrine and transforming growth factor type β
Authors: Houck Keith A. , Michalopoulos George K.
Published: February 2005

Norepinephrine (NE), acting through the α1‐adrenergic receptor, modulates the response of rat hepatocytes in primary culture to transforming growth factor type β1(TGFβ) by increasing the amount of TGFβ required for a given degree of inhibition of epidermal growth factor (EGF)‐induced DNA synthesis (Houck et al., J. Cell. Physiol.135:551–555, 1988). This effect was also found in hepatocytes isolated from regenerating livers but was greatly magnified in cells isolated between 12 and 18 hr after two‐thirds partial hepatectomy (PHX). During this period of enhanced sensitivity, NE was equally potent in terms of dose but more efficacious in the regenerating hepatocytes. As it did in control hepatocytes (Cruise et al., Science227:749–751, 1985), the α1‐adrenergic receptor mediated the activity of NE in regenerating hepatocytes. Vasopressin (VP) and angiotensin‐II (AG) also antagonized the effect of TGFβ and showed increased activity in regenerating hepatocytes but at only 50% or less of the maximal effect reached by NE. Regenerating hepatocytes isolated 24–72 hr after PHX exhibited decreased sensitivity to inhibition by TGFβ, with a nadir in 48‐hr‐regenerating cells. These findings suggest that NE may be involved in triggering the early phase of DNA synthesis during liver regeneration, with the subsequent acquisition of innate resistance to TGFβ responsible for continued proliferation at a time when TGFβ mRNA is known to be increasing in the liver (Braun et al., Proc. Natl. Acad. Sci. USA85:1539–1543, 1988). EGF induced increased DNA and protein synthesis in cultures of control hepatocytes; TGFβ inhibited the EGF‐induced DNA synthesis but had no effect on protein synthesis. This may be relevant to the latter stages of liver regeneration, when high levels of TGFβ mRNA are detected in liver and cellular hypertrophy predominates over hyperplasia.
Role of albumin arginyl sites in albumin‐induced reduction of endothelial hydraulic conductivity
Authors: Powers Michael R. , Blumenstock Frank A. , Cooper Jeffrey A. , Malik Asrar B.
Published: February 2005

We determined the effect of albumin on endothelial hydraulic conductivity (Lp) and the contributions of the positively charged arginyl and lysinyl residues of albumin in mediaing the effect. Studies were made using monolayers of cultured sheep pulmonary artery endothelial cells grown to confluence on polycarbonate filters. Water flux was measured as transendothelial hydrostatic pressure was varied from 5 to 20 cm H2 O. Lp was calculated from the slope of the relationship of water flux versus pressure. The Lp of endothelial monolayers perfused with albumin‐free Hanks Balanced Salt Solution (HBSS) was compared to perfusion with HBSS containing either native albumin, or albumin in which the arginyl residues were modified by a condensation reaction with 1,2‐cyclohexanedione (CHD‐albumin), or albumin in which the lysinyl residues were modified by a substitution reaction with succinic anhydride (SC‐albumin). Baseline Lp at 2.5 mg/ml native albumin was 1.6±0.1 × 10−6 cm/s/cm H2O compared to the filter Lp after removing cells of 3.0 ± 0.3 × 10−4 cm/s/cm H2O. Endothelial Lp increased by 60% when albumin concentration was decreased from 2.5 mg/ml to 0.5 mg/ml (P < 0.05), but did not change with an increase in concentration to 10 mg/ml. Albumin‐free buffer and CHD‐albumin increased endothelial Lp by 2.2 ± 0.3‐fold and 1.9 ± 0.3‐fold, respectively (P<0.05). All endothelial Lp values were restored to baseline when the native albumin concentration was returned to 2.5 mg/ml. Excess l‐arginine (2 × 10−3 M) inhibited the effect of native albumin and increased endothelial Lp 1.5 ± 0.02‐fold (P<0.05), but excess l‐lysine (4 × 10−3) in the presence of native albumin had no effect on Lp. None of the perfusates altered the filter Lp value. Neutral dextran (70 kD), in contrast to native albumin, had no effect on endothelial Lp. These results indicate that albumin reduces the hydraulic conductivity of endothelial monolayers in a concentration‐dependent fashion and that the arginyl residues of albumin are required for the response. The effect of albumin may be meditated by a charge interaction of albumin with the endo‐thelium.
Multiple sequential periods of DNA synthesis and quiescence in primary hepatocyte cultures maintained on the DMSO‐EGF on/off protocol
Authors: Chan Krammie , Kost Diane P. , Michalopoulos George
Published: February 2005

Repeated periods of DNA synthesis activity (each period consisting of two to three cycles) separated by intervals of quiescence in primary rat hepatocytes can be stimulated by sequential addition and removal of 2% dimethyl sulfoxide (DMSO) in the presence of epidermal growth factor (EGF). Hepatocytes can be kept in nonproliferating cultures for 7 days in media supplemented with 2% DMSO and EGF. If DMSO is removed while EGF is maintained, rat and human hepatocytes enter a 3 to 4 day period of DNA synthesis that declines rapidly by days 4 and 5. If DMSO is reintroduced into cultures at that point, kept on for 3 more days and removed again, hepatocytes reenter into proliferation with another self‐limited response of 3 to 4 days. Similar phenomena can seen with hepatocytes maintained in the presence of 3 mM phenobarbital. These protocols demonstrate that loss of responsiveness to mitogens in primary hepatocyte cultures is not an irreversible process. They also raise the possibility that signals for termination of DNA synthesis in hepatocytes emanate from hepatocytes themselves. These studies also suggest for the first time the possibility of designing in vitro systems that will allow clonal expansion of differential hepatocytes.
Receptor specific for certain nucleotides stimulates inositol phosphate metabolism and Ca2+ fluxes in A431 cells
Authors: Gonzalez Fernando A. , Alfonzo Ramona G. , Toro Jorge R. , Heppel Leon A.
Published: February 2005

We have recently reported that extracellular ATP induces a transient rise in cytosolic free Ca2+([Ca2+]i) in individual human epidermoid carcinoma A431 cells (Gonzalez et al: Journal of Cellular Physiology 135:269–276, 1988). We have now studied nucleotide specificity and desensitization for several early responses. Extracellular ATP (5–100 μM) caused the rapid formation of inositol trisphosphate and later its metabolites, inositol bisphosphate and inositol monophosphate. ATP also induced the efflux of 45Ca2+ from pre‐loaded cells. In addition, an increase in the rate of influx of 45Ca2+ stimulated by extracellular ATP was detected. Based on measurements of 45Ca2+ efflux and influx, desensitization studies, and chlortetracycline fluorimetry, we conclude that ATP mobilizes Ca2+ from internal stores and also stimulates entry across the plasma membrane. These effects were also displayed by UTP and to a lesser extent by ITP, while other nucleoside triphosphates as well as ADP, AMP, and adenosine, were inactive. Furthermore, desensitization of the response to ATP and UTP was seen after prolonged exposure to either nucleotide. This was specific for the nucleotide receptor since a response to bradykinin was not affected by the ATP pretreatment, although pretreatment with phorbol ester inhibited responses to both the nucleotides and bradykinin. Quantitative data on rate of recovery from the desensitized state and the response of desensitized cells to greatly elevated levels of ATP are presented. Extracellular ATP stimulated another early change previously reported for epidermal growth factor, namely, the phosphorylation of an 81‐kDa cytoskeletal protein. The stimulation of these events involves an ATP receptor whose properties differ from other ATP receptors that have been described.
Smooth muscle actin expression during P19 embryonal carcinoma differentiation in cell culture
Authors: Rudnicki Michael A. , Sawtell Nancy M. , Reuhl Kenneth R. , Berg Randy , Craig Jane C. , Jardine Karen , Lessard James L. , McBurney Michael W.
Published: February 2005

P19 embryonal carcinoma (EC) cells can be induced in vitro to differentiate into cells resembling those normally formed in the embryo. Among these cell types is one whose morphology is fibroblast‐like. Using indirect immunofluorescence and Western blot analysis with antibodies directed against various isoforms of actin, many of these fibroblast‐like cells were found to express smooth muscle actin isoforms. Northern blot analysis of RNA indicated the presence of a smooth muscle‐specific isoform of myosin heavy‐chain mRNA in immortal lines of these fibroblast‐like cells. These results suggest that these fibroblast‐like cells resemble fetal myofibroblastic or myoepithelial cells, which have a wide distribution during embryonic development.
Effects of interleukin 3 and of granulocyte‐macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue
Authors: Hattersley G. , Chambers T. J.
Published: February 2005

The effects of granulocyte‐macrophage colony stimulating factor (GM‐CSF), macrophage colony stimulating factor (M‐CSF), and interleukin 3 (IL3) on osteoclast formation were tested by incubation of murine hemopoietic cells on plastic coverslips and bone slices with GM‐CSF, M‐CSF, or IL3, with or without 1,25(OH)2 vitamin D3 (1,25(OH)2D3). Osteoclastic differentiation was detected after incubation by scanning electron microscopical examination of bone slices for evidence of osteoclastic excavations, and by autoradiographic assessment of cells for 1,25(OH)2D3‐calcitonin (CT) binding. The differentiation of CT‐receptor‐positive cells preceded bone resorption, but the number that developed correlated with the extent of bone resorption (r = 0.88). M‐CSF and GM‐CSF substantially reduced bone resorption and CT‐receptor‐positive cell formation. The degree of inhibition of bone resorption could not be attributed to effects on the function of mature cells, since M‐CSF inhibits resorption by such cells only by 50%, and GM‐CSF has no effect. GM‐CSF inhibited the development of mature function (bone resorption) to a greater extent than it inhibited CT‐receptor‐positive cell formation. Since CT‐receptor expression antedated resorptive function, this suggests that GM‐CSF resulted in the formation of reduced numbers of relatively immature osteoclasts. This suggests that it may exert a restraining effect on the maturation of cells undergoing osteoclastic differentiation in response to 1,25(OH)2D3. Conversely, IL3, which also has no effect on mature osteoclasts, by itself induced CT‐receptor expression but not bone resorption; in combination with 1,25(OH)2D3 it induced a threefold increase in bone resorption and CT‐receptor‐positive cells compared with cultures incubated with 1,25(OH)2D3 alone. IL3 did not induce CT‐receptors in peritoneal macrophages, blood mono‐cytes, or J 774 cells. The results suggest that IL3 induces only partial maturation of osteoclasts, which is augmented or completed by additional factors such as 1,25(OH)2D3.
Heat‐induced cytotoxicity in H2O2‐resistant Chinese hamster fibroblasts
Authors: Spitz Douglas R. , Li Gloria C.
Published: February 2005

Hydrogen‐peroxide‐resistant Chinese hamster fibroblasts, derived from the HA‐1 cell line, were isolated following continuous culturing in the presence of progressively increasing concentrations of hydrogen peroxide. The hydrogen‐peroxide‐resistant phenotype has been stable for over 360 days following removal from H2 O2 stress. These H2 O2‐resisant cell lines demonstrate increased resistance to hyperthermic cell killing, mediated by continuous heating at 43°C but not 45°C. The relationship between mammalian cellular adaptation to oxidative stress mediated by H2 O2 and resistance to 43°C hyperthermia is discussed.