bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for involvement of more than one class of glycoprotein in cell interactions with fibronectin
Authors: Urushihara Hideko , Yamada Kenneth M.
Published: February 2005

The receptor mechanism by which cells attach to fibronectin has been investigated by a combined immunologic and electrophoretic approach. One particular antiserum directed against 3T3 cell plasma membranes was found to contain antibodies that blocked spreading of these murine cells on fibronectin but not on laminin or serum spreading factor (vitronectin). Proteolysis experiments confirmed that this cell line has calcium‐protected polypeptides necessary for cell spreading on fibronectin. Consequently, protein antigens were fractionated according to size by SDS gel electrophoresis, and antigens that could block the inhibitory activity of the polyclonal antibody were identified. One class of blocking antigen appeared to correspond to the 140,000‐dalton complexes favored by several laboratories as fibronectin receptor candidates, but a second class of 45,000 daltons was also apparent. This 45,000‐dalton antigen was a major absorbing activity from 3T3 cell membranes and the predominant activity from L929 membranes. By isoelectric focusing and two‐dimensional gel electrophoresis, it was found to exist as a set of isoelectric point variants with pK = 5.3 to 6.2. Our results indicate that current models postulating a simple, unimolecular receptor mechanism for fibronectin may be oversimplified and that fibronectin may instead interact with more than one protein receptor component on the fibroblast cell surface.
Persistent production of colony‐stimulating factor (CSF‐1) by cloned bone marrow stromal cell line D2XRII after X‐irradiation
Authors: Naparstek Elizabeth , Donnelly Thomas , Shadduck Richard K. , Waheed Abdul , Wagner Katherine , Kase Kenneth R. , Greenberger Joel S.
Published: February 2005

The adherent stromal layer in long‐term bone marrow cultures (LTBMC) provides the cellular environment necessary for the in vitro proliferation and differentiation of pluripotential hematopoietic stem cells. The role of humoral hematopoietic growth factors, colony‐stimulating factors (CSF) in the regulation of hematopoietic cell production in this system is poorly understood. We have recently isolated and cloned an adherent cell line, D2XRII, derived from murine LTBMC. Plateau phase 25 cm2 cultures of 2 × 106 D2XRII cells in 8.0 ml produced CSF‐1 (M‐CSF) at around 100–150 units/0.1 ml medium. Following X‐irradiation there was a dose‐dependent decrease in the production of CSF‐1 to a plateau of 50% of control levels at 10,000 rad. Higher doses did not produce a further decrease. The X‐ray dose reducing CSF‐1 production to 50% was 100‐fold above the lethal dose as measured by clonagenic survival following trypsinization and replating. Trypsinized replated viable adherent but nondividing X‐irradiated D2XRII cells were maintained for up to 8 weeks after irradiation and demonstrated continuous production of CSF‐1. The data indicate significant divergence of two biologic effects of X‐irradiation on plateau‐phase marrow stromal cells: physiologic function of adherence and CSF‐1 production, versus proliferative integrity. This divergence of effects may be very relevant to understanding the mechanism of X‐irradiation‐associated marrow suppression and leukemogenesis.
EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells
Authors: MacLeod Carol L. , Luk Andrew , Castagnola Janice , Cronin Maureen , Mendelsohn John
Published: February 2005

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5–10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4‐day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF‐treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF‐treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF‐induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.
Volume changes in activated human neutrophils: The role of Na+/H+ exchange
Authors: Grinstein Sergio , Furuya Wendy , Cragoe Edward J.
Published: February 2005

The apparent volume of neutrophils, as measured electronically with the Coulter counter, has been reported to increase upon treatment with chemotactic factors. The occurrence of a volume change was confirmed by forward angle light scattering and by isotopic measurements of intracellular water space in cells treated with 12‐O‐tetradecanoylphorbol 13, acetate (TPA) or formyl‐methionyl‐leucyl‐phenylalanine (FMLP). Cell swelling was associated with an increase in the osmotic content of the cells, determined from Boyle‐van't Hoff plots, and with an increase in Na+ content, measured by flame photometry. The volume change was inhibited by replacement of extracellular Na+ with K+ or N‐methyl‐D‐glucamine+, or by addition of amiloride. Swelling was also inhibited by the 5‐N‐substituted analogs of amiloride, which are potent specific inhibitors of the Na+/H+ antiport. This pathway is activated in neutrophils by both TPA and FMLP. Activation of Na+/H+ exchange, determined as a Na+‐dependent and amiloride‐sensitive cytoplasmic alkalinization, was also found when neutrophils were treated with hypertonic solutions. The hypertonic activation of the antiport was similarly followed by cell swelling, detectable by electronic sizing. The results indicate that activation of Na+/H+ exchange can lead to significant cell swelling in neutrophils.
Hormonal control of the replication of human fetal fibroblasts: Role of somatomedin C/insulin‐like growth factor I
Authors: Conover Cheryl A. , Rosenfeld Ron G. , Hintz Raymond L.
Published: February 2005

Sparse cultures of fetal and postnatal human fibroblasts were equivalent in their responsiveness to the mitogenic action of somatomedin C/insulin‐like growth factor I (SM‐C/IGF‐I). At both developmental stages, the addition of SM‐C/IGF‐I (100 ng/ml) increased cell number at day 3 1.4‐fold in serum‐free medium and 2‐fold in the presence of 0.25% human hypopituitary serum. Furthermore, dose‐response curves indicated that there was no difference in the sensitivity of fetal and postnatal fibroblasts to the growth‐promoting effects of SM‐C/IGF‐I, with a half‐maximal response occuring at 6 ng/ml SM‐C/IGF I. This biological action of SM‐C/IGF‐I correlated with SM‐C/IGF‐I binding to fetal and postnatal fibroblast monolayers. Epidermal growth factor (EGF) and platelet‐derived growth factor (PDGF) also stimulated replication of fetal and postnatal fibroblasts. The mitogenic effects of SM‐C/IGF‐I, EGF, and PDGF were additive. Dexamethasone, which alone had no effect, was synergistic with SM‐C/IGF‐I in stimulating replication of postnatal fibroblasts. The combination of SM‐C/IGF‐I (100 ng/ml), dexamethasone (10−7 M), EGF (10 ng/ml), and PDGF (5 ng/ml) had the same mitogenic effectiveness as 10% calf serum (CS) in postnatal cells. In marked contrast, there was no mitogenic interaction between SM‐C/IGF‐I and dexamethasone in fetal fibroblasts. In fetal cells, SM‐C/IGF‐I + EGF + PDGF ± dexamethasone could only account for 50% of the activity of 10% CS. Moreover, fetal cells were 50–100% more responsive than postnatal cells to the proliferative effect of serum.
Removal of cellular water prevents the reformation of the interphase nucleus
Authors: Zieve Gary W. , Slitzky Barry E.
Published: February 2005

The mature snRNP (small nuclear ribonucleoprotein) particles are localized quantitatively in the interphase nucleus. Like many nuclear antigens, they distribute throughout the cytoplasm after the nuclear envelope breaks down during mitosis and then return to the newly formed daughter nuclei in early G1. Their abundance and stability and the availability of monoclonal antibodies that recognize them, make the snRNP particles a useful model system for studying the reformation of the nucleus at the completion of mitosis. A wide variety of metabolic inhibitors and alterations in normal culture conditions were investigated for their ability to interfere with the return of the snRNP particles to daughter nuclei after mitosis. None of the well‐characterized cytoskeletal inhibitors, biosynthetic inhibitors, calcium antagonists, nor ionophores were effective in interfering with this return. However, the removal of cellular water by exposure of cells to hypertonic medium during mitosis blocked the reformation of the nucleus and trapped the snRNP particles in the cytoplasm. In medium of twice the normal tonicity, the function of the mitotic spindle and the cleavage furrow are inhibited, however, the cells reattach to the substratum as if returning to interphase. The chromatin stays condensed and does not form a normal interphase nucleus and the snRNP particles stay dispersed throughout the cytoplasm. This condition is reversible and after return to normal medium the nucleus reforms and the snRNP particles collect in the new nuclei. After gentle extraction of metaphase cells, about 30% of the snRNP particles are soluble, however, the remainder are associated with an insoluble remnant. These data are consistent with the notion that the snRNP particles accumulate in the nucleus due to both preferential solubility and specific binding sites in the interphase nucleus.
Transformation of diploid human fibroblasts by DNA transfection with the v‐sis oncogene
Authors: Fry Dennis G. , Milam Lonnie D. , Maher Veronica M. , McCormick J. Justin
Published: February 2005

The simian sarcoma virus (SSV) oncogene (v‐sis) has a high degree of homology to the cellular gene coding for the B peptide of human platelet‐derived growth factor (PDGF), a potent fibroblast mitogen. The cellular homolog of v‐sis is activated in some mesenchymal human tumors and cell lines drived from them. To determine the phenotype produced by v‐sis in diploid human fibroblasts, we constructed plasmids containing the SSV provirus and drugresistance markers and transfected them into early‐passage human cells. Fibroblasts that had integrated the plasmid were selected for drug resistance and shown to contain and express the v‐sis oncogene by DNA and RNA hybridization. The v‐sis‐expressing cells grew to higher saturation densities than control cells transfected with vector plasmid alone and formed large, well defined foci. This allowed selection of transfectants directly for focus formation. The v‐sis transformed cells continued to grow well in the absence of serum, whereas age‐matched, vector‐transfected control cells ceased replicating under these conditions so that the final difference in density between the two populations was tenfold. Incorporation of thymidine in serum‐free medium by the v‐sis‐transformed cells was independent of exogenous PDGF. In contrast, PDGF increased thymidine incorporation in such medium by the control cells to the level found in the v‐sis‐transformed cells with or without added PDGF. These results suggest that expression of the v‐sis oncogene in diploid human fibroblasts causes sufficient endogenous synthesis of the B chain of PDGF to allow transformants to grow to abnormally high cell densities. When individual v‐sis‐transformed cells were grown on a background of normal cells, this higher cell density at confluence could be visualized as a focus.
Collagenase production by smooth muscle: Correlation of immunoreactive with functional enzyme in the myometrium
Authors: Blair Harry C. , Teitelbaum Steven L. , Ehlich Lynn S. , Jeffrey John J.
Published: February 2005

A monospecific antibody to rat uterine collagenase has been produced and employed to study the cell of origin and the time course of production of this enzyme in the involuting rat uterus. The specificity of the anti‐collagenase antibody was confirmed by immunoprecipitation, Western analysis, and by its ability to inhibit the activity of collagenase. Parallel measurements of functional enzyme, both latent and active, bound to tissue collagen were also made in nonpregnant, gravid, and postpartum rat uteri. Immunohistochemical staining of collagenase in sections of rat uterus showed the enzyme to be present in the perinuclear region of the smooth muscle cells only of the involuting myometrium. No detectable collagenase was present in the prepartum or nonpregnant uterus. Identity of the smooth muscle cells was confirmed using an anti‐smooth muscle actin antibody. In addition, the cultured uterine cells from which the immunizing antigen was obtained were also identified as smooth muscle cells. Specificity of the tissue staining was confirmed by the ability of pure rat uterine collagenase to block the reaction of the antibody with the tissue. These observations indicate that smooth muscle cells are capable of producing collagenase and are consistent with the hypothesis that this enzyme presides over the massive collagen degradation seen in postpartum uterine involution. Furthermore, measurement of collagenase bound to uterine collagen revealed that collagenase activity could be detected only at the time that the cells could be seen to be producing the enzyme by immunolocalization. These findings support the concept that collagenase is produced only as needed and not stored, either intra‐ or extracellularly.
Production of C5a antagonist by synovial and peritoneal tissue fibroblasts
Authors: Matzner Yaacov , Gavison Rivka , Shlomai Zipora , Ben‐Bassat Hanna , Liebergal Meir , Robinson Dwight R. , Babior Bernard M.
Published: February 2005

Fibroblasts grown from synovial and peritoneal tissues release into the medium an inhibitor of neutrophil chemotaxis. The inhibitor resembles the antagonist previously described in synovial and peritoneal fluids. It is a heat stable (56°C) protein of MW ∼40 kDa that counteracts the chemotactic activity of zymosan‐activated serum or purified C5a but not the peptide chemoattractant F‐met‐leu‐phe. No chemotactic inhibitor was detected in media from skin fibroblast cultures or in formal human sera. It is suggested that the inhibitor is produced locally by synovial and peritoneal fibroblasts and that it might play a role in the regulation of inflammation at sites lined with mesothelium.
Restricted growth of rat kidney proximal tubule cells cultured in serum‐supplemented and defined media
Authors: Miller John H.
Published: February 2005

Proximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll‐gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum‐containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen‐coating of the dishes nor addition of the differentiation inducer hexamethylene‐bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum‐supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell.
Ca2+‐dependent cell surface protein phosphorylation may be involved in the initiation of DNA synthesis
Authors: Kleine Leonard P. , Whitfield James F. , Boynton Alton L.
Published: February 2005

Incubating T51B rat liver cells in Ca2+‐deficient, serum‐rich medium containing only 0.02 mM Ca2+ strikingly decreased the phosphorylation of several trypsin‐removable cell surface proteins and arrested the cells in late G1 phase. Raising the Ca2+ concentration in the Ca2+ ‐deficient medium from 0.02 mM to 0.5 mM or adding 80 nM TPA (12‐O‐tetradecanoyl‐phorbol‐13‐acetate), a, protein kinase C activator, stimulated the phosphorylation of a certain set of surface proteinS'Within 5 min and the initiation of DNA replication within the next 2 hr. By contrast, incubation in the same Ca2+ ‐deficient medium, which does not affect the proliferation of neoplastic T51B‐261B cells, did not reduce the phosphorylation of cell surface proteins. These observations suggest that the stimulation of a Ca2+ ‐dependent protein kinase (possibly protein kinase C) directly or indirectly phosphorylates certain cell surface proteins that might be part of the mechanism that triggers the Ca2+‐dependent G1→S transition of normal cells. They also suggest that an alteration of this Ca2+‐dependent protein kinase might be the reason for neoplastic cells being able to proliferate in the face of an external Ca2+ shortage that would stop the proliferation of normal cells.
Spermidine mediates degradation of ornithine decarboxylase by a non‐lysosomal, ubiquitin‐independent mechanism
Authors: Glass James R. , Gerner Eugene W.
Published: February 2005

The mechanism of spermidine‐induced ornithine decarboxylase (ODC, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum‐free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine (Glass and Gerner: Biochem. J., 236:351–357, 1986; Sertich et al.: J. Cell Physiol., 127:114–120, 1986). Treatment of cells with 10 μM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [35S]methionine‐labeled ODC and separation by two‐dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine‐mediated inactivation of ODC occurred in a temperature‐dependent manner exhibiting pseudo‐firstorder kinetics over a temperature range of 22–37°C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22°C for 3 hours with 10 μM spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents (NH4Cl, chloroquine, antipain, leupeptin, chymostatin) had no effect on ODC degradation. ODC turnover was not dependent on ubiquitin‐dependent proteolysis. Shift of ts85 cells, a temperature‐sensitive mutant for ubiquitin conjugation, to 39°C (nonpermissive for ubiquitin‐dependent proteolysis) followed by addition of spermidine led to a rapid decline in ODC activity, with a rate similar to that seen at 32°C (the permissive temperature). In contrast, spermidine‐mediated ODC degradation was substantially decreased by inhibitors of protein synthesis (cycloheximide, emetine, and puromycin). These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non‐lysosomal, ubiquitin‐independent pathway.
Graded amplification of the Na, K‐ATPase across a subclonal series: Effects on membrane physiology
Authors: Pauw Peter G. , Ash John Frederick
Published: February 2005

We have generated a series of clonally related cell lines which differ in the level of amplified expression of the Na, K‐ATPase. These lines, originally derived from the ouabain resistant HeLa variant C+, expressed different numbers of binding sites for the Na, K‐ATPase inhibitor ouabain, ranging from 2.9 × 106/cell to 11.8 × 106/cell. Amplification of the genes for both subunits of the enzyme was also seen but was not strictly correlated with level of expression. The influxes of histidine and tetraphenylphosphonium were measured across a series, including HeLa S3 and revertants, expressing from 0.74 × 106 to 10.5 × 106 ouabain‐binding sites per cell. Tetraphenylphosphonium influx rate, presumed to be a function of membrane potential, varied linearly with ouabain binding site number, while histidine influx varied with the log of ouabain binding site number. Our results suggest that membrane potential increases in a simple fashion across our series of amplified lines. However, histidine influx was unaffected by treatments which cause membrane depolarization and a decrease in tetraphenylphosphonium influx rate. We propose that increasing histidine influx rates across our amplified series reflects exchange acceleration of L system transport due to increased intracellular pools of L system reactive amino acids.

The Na, K‐ATPase is ultimately responsible for most active transport across the plasma membrane. The consistent, graded physiological alterations seen across this series of closely related lines, chosen for graded enzyme expression, demonstrate the value of this novel genetic approach to the study of the energization of membrane transport.
Phorbol esters and mitogenesis: Comparison of the proliferative response of parental and Na+K+Cl−‐contransport‐defective BALB/c 3T3 cells to 12‐0‐tetradecanoylphorbol‐13‐acetate
Authors: O'Brien Thomas G. , Prettyman Ralph
Published: February 2005

The ability of the phorbol ester 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) to stimulate the growth of quiescent BALB/c 3T3 cell lines lacking Na+K+Cl− contransport activity was tested. We have previously isolated and characterized two mutant cell lines defective in this important ion transport system by mutagenesis and selection in medium containing low K+. To test our hypothesis that loss of this transport activity might abrogate the proliferative response to TPA, two kinds of mitogenesis assays were performed. First, the effect of 0.16 μM TPA on the saturation density of parental vs. mutant cell lines was determined. TPA caused a small but reproducible 30–35% increase in the saturation density of mutant cells compared to the 100–120% increase seen in parental cell lines. Second, the effect of TPA on the incorporation of 3H‐thymidine into cell nuclei (labeling index) was measured. While some variability from experiment to experiment in the extent and time course of the response of mutant cells was noted, TPA either had no effect or only a small effect on the labeling index when compared to the response of parental cells. When a range of concentrations of TPA (0.016–1.6 μM) was tested, neither cell line exhibited a large response to any concentration. These results suggest that loss of Na+K+Cl− contransport activity decreases the response of these cells to the mitogenic action of TPA.
Hydrogen peroxide or heat shock induces resistance to hydrogen peroxide in Chinese hamster fibroblasts
Authors: Spitz Douglas R. , Dewey W. C. , Li Gloria C.
Published: February 2005

Survival after H2O2 exposure or heat shock of asynchronous Chinese hamster ovary cells (HA‐1) was assayed following pretreatment with mildly toxic doses of either H2O2 or hyperthermia. H2O2 cytotoxicity at 37°C, expressed as a function of mM H2O2 was found to be dependent on cell density at the time of treatment. The density dependence reflected the ability of cells to reduce the effectiveness of H2O2 as a cytotoxic agent. When the survival data were plotted as a function of μmoles H2O2/cell at the beginning of the treatment, survival was independent of cell density. Cells pretreated with 0.1 mM (3–5 μmoles/cell × 10−7) H2O2 for 1 hr at 37°C (30–50% survival) became resistant to a subsequent H2O2 treatment 16–36 hr after pretreatment [dose modifying factor (DMF) at 1% isosurvival = 4–6]. Their resistance to 43°C heating, however, was only slightly increased over controls 16–36 hr following pretreatment (DMF at 1% isosurvival = 1.2). During this same interval, the synthesis of protein migrating in the 70 kD region of a one‐dimensional SDS‐polyacrylamide gel was enhanced twofold in the H2O2‐pretreated cells. When the cells were heated for 15 min at 45°C (40–60% survival), the survivors became extremely resistant to 43°C heating and somewhat resistant to H2O2 (DMF at 1% isosurvival = 2). The heat‐induced resistance to heat developed much more rapidly (reached a maximum between 6 and 13 hr) following pretreatment than the heat‐induced rsistance to H2O2 (16–36 hr). The enhanced synthesis of 70 kD protein after heat shock was greater in magnitude and occurred more rapidly following preheating than following H2O2 pretreatment. The cells that became resistant to H2O2 by either pretreatment (H2O2 or heat shock) also increased their ability to reduce the H2O2 cytotoxicity from the treatment medium beyond that of the untreated HA‐1 cells. This may be one of the mechanisms involved in the increased resistance and a common adaptive mechanism induced by both stresses. These data indicate that mammalian cells develop resistance to H2O2 following mild pretreatment with H2O2 or heat shock. The cross‐resistance induced by H2O2 and heat shock reinforce the hypothesis that some overlap in mechanisms exist between the cellular responses to these two stresses. However, the failure of H2O2 pretreatment to induce much resistance to heat indicates that there are also differences in the actions of the two agents.
Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP
Authors: Boonstra Johannes , Mummery Christine L. , Feyen Alie , de Hoog Wouter J. , van der Saag Paul T. , de Laat Siegfried W.
Published: February 2005

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high‐affinity binding sites and a small increase in the number of low‐affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF‐receptor phosphotyrosine kinase activity. These results indicate that neither EGF‐binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.
Membrane potential and cation content of osteoblast‐like cells (UMR 106) assessed by fluorescent dyes
Authors: Civitelli Roberto , Reid Ian R. , Halstead Linda R. , Avioli Louis V. , Hruska Keith A.
Published: February 2005

A number of cellular functions have recently been associated with alterations of the membrane potential in non‐excitable cells. To assess the electrophysiologic regulation of osteoblast function, a method for measuring the membrane potential (Em) of a rat osteogenic sarcoma cell line (UMR 106) by the voltage‐sensitive oxonol dye di‐BA‐C4(3) was developed. The fluorescent signal of di‐BA‐C4(3) was calibrated through a null point method using the protonophore FCCP. At null point, Em is equivalent to H+ equilibrium potential, and may be calculated by the Nernst equation. Intracellular pH (pHi) changes induced by the protonophore were monitored using BCECF, a pH‐sensitive fluorescent probe. In the presence of FCCP, intracellular pH was found to be linearly correlated to extracellular pH (pHo). Therefore, the value of pHi at null point was extrapolated as well. With this technique, we estimated the plasma membrane potential of the “putative” rat osteoblasts (UMR 106) as ‐ 28.3 ± 4.0 mV (n = 10). This method corrected the 16% overestimation of Em derived from the assumption that pHi does not change during the calibration procedure, as described in previous studies employing pH null point techniques. With null point methods, using BCECF and the carboxylic ionophores nigericin and monensin, intracellular concentrations of potassium and sodium were also measured and found to be 125 ± 0.7 mM (n = 3) and 24 ± 5.3 mM (n = 3), respectively. Although the Em of UMR 106 cells was dependent on extracellular potassium concentration, these cells did not behave as a potassium electrode. The sodium/potassium permeability ratio, calculated by the Goldman equation, was estimated at 0.317. This high membrane permeability to sodium may contribute to the genesis of the low plasma membrane potential of UMR 106 cells.
Molecular sieving characteristics of the cultured endothelial monolayer
Authors: Siflinger‐Birnboim Alma , del Vecchio Peter J. , Cooper Jeffrey A. , Blumenstock Frank A. , Shepard Jonathan M. , Malik Asrar B.
Published: February 2005

We examined the selectivity of the bovine pulmonary artery endothelial monolayer in vitro to molecules of different sizes. The cultured bovine pulmonary endothelial monolayer was grown on a gelatinized filter and the transendothelial transport was studied by determining the permeability of molecules ranging from 182 to 340,000 daltons under diffusion conditions. The permeabilities across the cultured bovine endothelium were modeled according to cylindrical pore theory. The data were best fit by a two‐pore model with radii 65 Å and 304 Å and a ratio of small to large pores of 160:1. The results indicate that the cultured endothelial monolayer is a selective barrier to molecules of different sizes and that the molecular selectivity is consistent with a diffusional pathway through endothelial pore equivalents. The cultured endothelial monolayer is a useful system for studying the permeability characteristics of the endothelial barrier.
Influence of hormone and growth factor interactions on the proliferative potential of normal rat mammary epithelial cells in vitro
Authors: Ethier Stephen P. , Kudla Arthur , Cundiff Kimberly C.
Published: February 2005

These experiments were aimed at using a recently developed serum‐free culture system for growth of normal rat mammary epithelial (RME) cells in vitro to examine the interactions of specific hormones and growth factors on the proliferative potential of these cells. RME cells were obtained by enzymatic dissociation of mammary tissues of Lewis rats. Primary cultures were started by plating 2 × 105 RME cells per 60‐mm type I collagen‐coated tissue culture dish. Cultures were maintained in a basal medium that consisted of Ham's F‐12 medium supplemented with bovine serum albumin (BSA), ethanolamine (EA), and transferrin (Tf), which, by itself, did not support RME cell proliferation. Insulin (I), hydrocortisone (HC), and epidermal growth factor (EGF), when added to the basal medium interacted synergistically to stimulate RME cell proliferation, but this effect was dependent on the additional presence of cholera toxin (CT). Under these conditions a greater‐than‐tenfold increase in cell number over a 10‐day culture period was obtained. Insulin could be replaced by physiological levels of insulin‐like growth factor‐I (IGF‐I). CT could be replaced by other agents that elevate intracellular levels of cyclic adenosine 3′:5′ monophosphate (cAMP) such as dibutyryl‐cAMP (db‐cAMP), prostaglandin E1 (PGE‐1), and/or isobutylmethylxanthine (IBMX). Prolactin (M) or progesterone (P) potentiated the effect of I, HC, EGF, and CT, resulting in an additional twofold increase in cell number over that found in their absence. However, addition of both hormones was no more effective than either one alone. Furthermore, addition of M or P in the absence of EGF had no effect on RME cell proliferation. Addition of 17‐B‐estradiol (E2) to the I‐, HC‐, EGF‐, and CT‐containing medium also resulted in enhanced RME cell proliferation. These results point to a number of hormone and growth factor interactions that influence the proliferation of normal RME cells in vitro.
Stimulation by serum of the Na+/H+ antiporter in quiescent pig kidney epithelial (LLC‐PK1) cells and role of the antiporter in the reinitiation of DNA synthesis
Authors: Haggerty John G. , Agarwal Neeraj , Amsler Kurt , Slayman Carolyn W. , Adelberg Edward A.
Published: February 2005

LLC‐PK1 cells can be brought into a classical quiescent state by depriving them of serum for 6 days. At this time, pulse‐labeling with [3H]‐thymidine shows that only 3% of the cells are synthesizing DNA, but the quiescent cells can be stimulated with serum to re‐enter the cell cycle at a point early in G1. The rate of amiloride‐sensitive 22Na+ uptake (as a measure of the Na+/H+ antiporter) is relatively low during quiescence; it rises 2‐ to 3‐fold within 4 h after serum addition. This increase in antiporter activity appears to be required for the resumption of DNA synthesis in the absence of bicarbonate, because ethylisopropylamiloride (EIPA) blocks [3H]‐thymidine incorporation when serum is added to cells in bicarbonate‐free medium. In the presence of bicarbonate, however, EIPA has no effect on [3H]‐thymidine incorporation, indicating that another (bicarbonate‐dependent) transport system can substitute for the antiporter under these conditions.
Verapamil inhibits serotonin uptake of endothelial cells in culture by a mechanism unrelated to Ca2+ channel blockade
Authors: Lee S.‐L. , Long K. , Ueda S. , Fanburg B. L.
Published: February 2005

Verapamil inhibited Na+‐dependent uptake of serotonin (5‐HT) by bovine pulmonary artery endothelial cells in culture both exposed to room air and stimulated by prior exposure to anoxia. The effect of verapamil occurred even in the absence of Ca2+ from the assay medium. Although absence of Ca2+ from the medium moderately reduced 5‐HT uptake, stimulation of uptake was nevertheless observed for cells previously exposed to anoxia. Verapamil altered the Km, but not the Vmax, of 5‐HT uptake. There was no change in 45Ca2+ uptake or release by cells previously exposed to anoxia as compared to those exposed to room air and verapamil did not influence 45Ca2+ fluxes by either set of cells. It is concluded that verapamil inhibits 5‐HT uptake by endothelial cells through a mechanism other than Ca2+ channel blockade; the results are consistent with competitive inhibition of a 5‐HT carrier. The stimulatory effect of anoxia on 5‐HT uptake does not occur through a change in Ca2+ fluxes.
Serum and α2‐macroglobulin induce transient hyperpolarizations in the membrane potential of an osteoblastlike clone
Authors: Dixon S. J. , Aubin J. E.
Published: February 2005

Using microelectrode techniques, we have observed that the application of serum or α2‐macroglobulin (α2M) induces transient hyperpolarizations in the membrane potential of a rat osteosarcoma clone (ROS 17/2). Hyperpolarizations arose from activation of Ca2+‐dependent K+ channels by transient increases in the concentration of intracellular free Ca2+. Hyperpolarizing spikes were observed for several h following the addition of fetal bovine serum (FBS) to cell cultures. Application of small volumes of FBS or α2M rapidly induced synchronized bursts of hyperpolarizing spikes. No response was elicited by serum‐free medium, latex beads, or bovine serum albumin (BSA). Immunofluorescence labeling patterns were consistent with the receptor‐mediated endocytosis of α2M but not BSA. The ligand specificity and kinetics of these hyperpolarizations suggest that they are associated with a receptor‐mediated event, possibly an early stage of receptor‐mediated endocytosis.
Relationship between pH, sodium, and shape changes in chemotactic‐factor‐stimulated human neutrophils
Authors: Faucher N. , Naccache P. H.
Published: February 2005

The relationship between the chemotactic‐factor‐elicited changes in the intracellular pH and the shape of human neutrophils was investigated using simultaneous measurements of both parameters. The results demonstrate first that fMet‐Leu‐Phe and leukotriene B4 elicit qualitatively similar pH and shape change responses from the neutrophils. A relationship between the chemoattractant‐elicited decrease in cytoplasmic pH and the shape changes is indicated by several findings including: (1) the similarities in the time courses of the two responses, (2) the ability of propionic acid to induce a transient and pertussis‐toxin‐sensitive shape change response, and (3) the ability of the calcium ionophore A23187 to similarly induce both responses under conditions when the degranulation is minimized. On the other hand, several other results indicate that the drop in pH is not a sufficient condition for the chemotactic‐factor‐stimulated shape changes. These include: (1) the ability of pertussis toxin to inhibit the shape changes induced by propionic acid and by A23187 without affecting the drop in pH, and (2) the observation that the drop in pH induced by propionic acid persists significantly longer than the shape change. Increasing the cytoplasmic pH by adding ammonium chloride was also found to cause shape changes in the neutrophils. The response to the base differs in two important aspects from that caused by proprionic acid: it is pertussis‐toxin‐insensitive, and it is long‐lived. Chemotactic factors have been found to induce a shape change under conditions when the internal pH was artificially increased or decreased, indicating that it is not the absolute cytoplasmic pH that represents the internal signalling parameter. The results are discussed in terms of the activation of the cytoskeletal network of the neutrophils by chemotactic factors.
Retrovirus‐induced feline pure red cell aplasia: The kinetics of erythroid marrow failure
Authors: Abkowitz Janis L. , Holly Richard D. , Adamson John W.
Published: February 2005

Cats viremic with feline leukemia virus subgroup C (FeLV‐C) develop pure red cell aplasia (PRCA) characterized by the loss of detectable late erythroid progenitors (CFU‐E) in marrow culture. Normal numbers of early erythroid progenitors (BFU‐E) and granulocyte‐macrophage progenitors (CFU‐GM) remain, suggesting that the maturation of BFU‐E to CFU‐E is impaired in vivo. We have examined the cell cycle kinetics of BFU‐E and their response to hematopoietic growth factor(s) to better characterize erythropoiesis as anemia develops. Within 3 weeks of FeLV‐C infection, yet 6–42 weeks before anemia, the fraction of BFU‐E in DNA synthesis as determined by tritiated thymidine suicide increased to 43 ± 4% (normal 23 ± 2%) while there was no change in the cell cycle kinetics of CFU‐GM. In additional studies, we evaluated the response of marrow to the hematopoietic growth factor(s) present in medium conditioned by FeLV‐infected feline embryonic fibroblasts (FEA/FeLV CM). With cells from normal cats or cats viremic with FeLV‐C but not anemic, a 4‐fold increase in erythroid bursts was seen in cultures with 5% FEA/FeLV CM when compared to cultures without CM. However, just prior to the onset of anemia, when the numbers of detectable CFU‐E decreased, BFU‐E no longer responded to FEA/FeLV CM in vitro. BFU‐E from anemic cats also required 10% cat or human serum for optimal in vitro growth. These altered kinetics and in vitro growth characteristics may relate to the in vivo block of BFU‐E differentiation and PRCA. Finally, when marrow from cats with PRCA was placed in suspension culture for 2 to 4 days in the presence of cat serum and CM, the numbers of BFU‐E increased 2‐ to 4‐fold although no CFU‐E were generated. By 4 to 7 days, CFU‐E were detected, suggesting that conditions contributing to the block of erythroid maturation did not persist. The suspension culture technique provides an approach to study further the defect in erythroid differentiation characteristic of feline PRCA.
Receptor binding and mitogenic effects of insulin and insulinlike growth factors I and II for human myeloid leukemic cells
Authors: Pepe Michael G. , Ginzton Nancy H. , Lee Philip D. K. , Hintz Raymond L. , Greenberg Peter L.
Published: February 2005

Insulin and insulinlike growth factors I and II (IGF‐I and IGF‐II) influence mesodermal cell proliferation and differentiation. As multiple growth factors are involved in hemopoietic cell proliferation and differentiation, we assessed the receptor binding and mitogenic effects of these peptides on a panel of mesodermally derived human myeloid leukemic cell lines. The promyelocytic cell line HL60 had the highest level of specific binding for these 125I‐labeled ligands, with lower binding to the less differentiated myeloblast cell line KG1 and undifferentiated blast variants of these cell lines (HL60blast, KG1a). Insulin binding affinity and receptor numbers were reduced significantly by chemically induced granulocytic differentiation of HL60 cells and was unchanged following induced monocytic differentiation. No substantial alteration in IGF‐I or ‐II binding occurred with induced HL60 cell differentiation. Insulin and IGF‐I demonstrated cross competition for receptor binding and down‐regulated their homologous receptors without detectable cross modulation of the heterologous receptors on HL60 cells. IGF‐I and insulin increased HL60 cell proliferation, as assessed by 3H‐thymidine uptake, IGF‐I> insulin. IGF‐I binding and mitogenic effects were blocked by the monoclonal anti‐IGF‐I receptor antibody IR3, indicating that IGF‐I‐induced proliferative effects were mediated via its homologous receptor. In contrast, insulin binding and mitogenesis displayed blocking by both anti‐IGI‐I and anti‐insulin receptor antibodies, indicating mediation of its activity through both receptors. These data demonstrate specific binding and mitogenic interactions between insulin, IGFs, and hemopoietic cells which are associated with their state of differentiation.
Effect of hypertonicity on hexose transporter regulation in chicken embryo fibroblasts
Authors: Tillotson Loyal G. , Isselbacher Kurt J.
Published: February 2005

The regulation of hexose transporters of cultured fibroblasts was investigated by exposing chicken embryo fibroblasts (CEF) to hypertonic culture medium, a condition known to enhance hexose transport activity. The effects of hypertonicity and the role of protein synthesis were examined with CEF in the basal (glucose fed) and transport enhanced (glucose starved) states. Glucose‐fed CEF exposed to hypertonic conditions developed four‐fold enhancement of hexose transport activity within 4 hrs; this declined in the following 20 hrs to a level slightly higher than the fed control. Protein synthesis was required in part for this effect, since the presence of cycloheximide during hypertonic exposure of fed CEF blocked the increase in of transport by almost 50%. Although the increased transport produced by glucose starvation was not further enhanced by hypertonicity, hypertonic treatment of starved CEF during glucose refeeding largely prevented the loss of transport acitivity to the basal, fed state. The hypertonic effects were concentration dependent (240mOsm optimal) and could be elicited with NaCl, KCl, or sucrose. Hypertonic treatment typically led to a greater than 50% decline in the incorporation of [3H]leucine into acid‐insoluble fractions. The changes in transport were evident at the plasma membrane level, and studies of membrane vesicles prepared from hypertonically treated fed CEF showed a doubling of both [3H]cytochalasin B binding and the Vmax of D‐glucose transport. These findings indicate that exposure of CEF to hypertonic conditions had some effects similar to those produced by glucose starvation and suggest that protein synthesis is to some extent involved in the regulation of hexose transporters in CEF.