bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of sodium butyrate on actin distribution in rat 3Y1 fibroblasts in monolayer culture
Authors: Yamada Koji , Sasaki Masafumi , Kimura Genki
Published: February 2005

We studied the effect of sodium butyrate, a potent G1/G2‐arresting agent, on actin distribution in rat 3Y1 fibroblasts in monolayer culture by fluorescence microscopy of cells stained with 7‐nitrobenz‐2‐oxa‐1, 3‐diazole phallacidine (NBD‐Ph). When randomly proliferating cells were arrested mainly in G1 phase with butyrate, a reversible overaccumulation of cellular net protein occurred. In the G1‐arrested cells, actin markedly accumulated at the margin of cells, and a network structure of actin stress fibers appeared. When density‐arrested cells were replated sparsely and rearrested in the G1, early S, and G2 phases with butyrate or hydroxyurea, the actin network was observed extensively in the cells arrested in the G1 and G2 phases with butyrate. These results agree with our previous results indicating the existence of some physiological similarity between cells in the G1 and G2 phases and suggest that actin distribution somehow depends on the phases of the cell cycle. The actin profiles observed by the NBD‐Ph staining were confirmed by transmission electronmicroscopy (TEM) of negatively stained whole cells. TEM further revealed that electron‐dense amorphous materials were present at crossing points in the network but rarely present on interconnecting microfilament bundles.
Differential induction of glucose‐regulated and heat shock proteins: Effects of pH and sulfhydryl‐reducing agents on chicken embryo cells
Authors: Whelan Sandra A. , Hightower Lawrence E.
Published: February 2005

Glucose‐regulated and heat shock proteins are two subsets of eukaryotic stress proteins that can be induced differentially, simultaneously, and reciprocally. Two new inducers, low extracellular pH and 2‐mercaptoethanol, that stimulate chicken embryo cells to synthesize glucose‐regulated proteins rapidly were found. Two classes of cellular targets for mercaptoethanol were defined operationally, one dependent on and the other independent of protein synthesis. A new inducer of heat shock proteins, high extracellular pH, was found as well. Inductions by low and high extracellular pH were inhibited by actinomycin D but were insensitive to cycloheximide. Inductions of glucose‐regulated and heat shock proteins are discussed in terms of changes in intracellular pH and sulfhydryl oxidation states.
Effect of cycloheximide and growth factors on gene expression in quiescent mouse embryo fibroblasts
Authors: Subramaniam M. , Shanmugam G.
Published: February 2005

Gene expression in quiescent mouse embryo fibroblasts was studied by labelling the cells with [14C] amino acids and analysing the proteins by electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate. Cycloheximide (CH) pretreatment of the cells was found to induce the synthesis of four proteins of molecular weights 72,000, 68,000, 42,000, and 29,000. These proteins were induced by CH both in serum‐arrested and serum‐ stimulated cells. Addition of platelet‐derived growth factor to serum‐arrested quiescent cells also induced the synthesis of these proteins. Addition of CH and fetal calf serum (20%) to quiescent cells resulted in a dramatic increase in the synthesis of actin and another protein of molecular weight 29,000. The 29,000‐dalton protein was present in higher quantities in the nuclei of induced cells. This protein appeared to be an early protein whose synthesis was transiently induced in quiescent cells within 3 hours of addition of 20% fetal calf serum (FCS). The synthesis of this protein was virtually turned off at 5‐6 hours after the addition of serum. However, if CH or a combination of CH and FCS was present, a continuous synthesis of the 29 K protein was observed.
Hydrogen peroxide toxicity may be enhanced by heat shock gene induction in Drosophila
Authors: Love Jack D. , Vivino Alfred A. , Minton Kenneth W.
Published: February 2005

Recent evidence suggests that low dose exposure of cells to hydrogen peroxide and/or induction of heat shock protein (HSP) synthesis will render cells resistant to the lethal effects of a subsequent high dose hydrogen peroxide stress. We explored this possibility in the Drosophila melanogasterSchneider tissue culture line 2. It was found that chronic low dose exposure (1 mM H2O2for 3 days) resulted in marked potentiation of the toxic effects of a subsequent high dose exposure (50 mM H2O2 for 1 h), as assessed by impairment of uridine incorporation and cell proliferation. Cells preexposed to low dose H2O2 exhibited enhanced heat shock gene transcription upon exposure to high dose H2O2, as compared to cells that did not receive low dose preexposure. Transcriptional induction of the heat shock genes by a mild non‐toxic heat shock resulted in marked enhancement of the anti‐proliferative effects of a subsequent H2O2 exposure. Thus, low dose hydrogen peroxide exposure or mild heating results in subsequent enhancement of high dose hydrogen peroxide toxicity; this effect correlates with enhanced heat shock gene expression. Possible mechanisms are discussed.
Effects of maturational agents on expression and secretion of two partially characterized high molecular weight milk‐related glycoproteins in MCF‐7 breast carcinoma cells
Authors: Abe Miyako , Kufe Donald
Published: February 2005

We have previously defined a human mammary epithelial antigen using a murine monoclonal antibody (MAb), designated DF3, prepared against a membrane‐enriched fraction of a human breast carcinoma. MAb DF3 detects a cell surface antigen with a molecular weight (mw) of approximately 300 Kd and a higher mw species also detectable in human milk. These findings and the demonstration that butyric acid (BA) increases DF3 antigen expression suggested that MAb DF3 reacts with a differentiation antigen detectable in human breast carcinoma cells. The results of the present study demonstrate that MAb DF3 reacts with two mucin‐like high mw glycoproteins (330 and 450 Kd) present in MCF‐7 breast carcinoma cells. The results also demonstrate that the intracellular content and secretion of DF3 antigen is increased by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and 1‐β‐D‐arabinofuranosylcytosine (ara‐C). Other known inducers of differentiation including retinoic acid (RA), hexamethylene bisacetamide (HMBA), 1,25‐dihydroxy vitamin D3 (1,25(OH)2D3) and certain polar solvents decrease DF3 antigen expression. Furthermore, the results demonstrate that DF3 antigen is secreted and that the extent coincides with changes in intracellular content. Finally, actinomycin D and cycloheximide inhibit the increases in DF3 antigen expression following TPA treatment thus suggesting that newly synthesized RNA and protein are required for induction of this antigen. Thus, the monitoring of DF3 antigen expression may provide a marker for studying maturation of human breast cancer cells.
Altered endocytosis in a mutant of LM fibroblasts defective in cell–cell fusion
Authors: Li Wande , Ryser Hugues J.‐P. , Shen Wei‐Chiang
Published: February 2005

Mutants of LM fibroblasts selected for their decreased ability to undergo polyethylene glycol‐induced cell‐to‐cell fusion (F40 subline) were examined for possible alterations of their ability to carry out endocytosis. Both fluidphase endocytosis of inulin and horseradish peroxidase and nonreceptor mediated adsorptive endocytosis of poly(L‐lysine) were reduced to 60% of control values. Comparable results were obtained when the uptake of poly(L‐lysine) was measured as internalization of surface‐bound label in label‐free medium or following continuous exposure. Accelerated breakdown of internalized label was ruled out as a cause for decreased label accumulation. Accelerated exocytosis is an unlikely cause, and it is suggested that the decreased uptake is due to a decrease in the constitutive membrane vesiculation process that leads to the formation of endocytotic vesicles. The capacity of F40 cells to degrade internalized horseradish peroxidase and poly(L‐lysine) was not impaired, nor was their susceptibility to the cytotoxic action of methotrexate‐poly(L‐lysine). This drug conjugate must be degraded inside cells and release small molecular methotrexate in order to be cytocidal. These data suggest that only the first step of nonspecific endocytosis is impaired, while the subsequent steps that require fusion of endosomes to lysosomes proceed normally. Since the formation of primary endosomes requires membrane fusion through the external aspect of the plasma membrane and in that respect resembles cell‐cell fusion, we propose the hypothesis that the observed decrease in endocytosis is related to the decreased ability of F40 cells to fuse with each other, and reflects a decreased efficiency of fusion processes at the external face of the plasma membrane.
Highly adherent mutants of SV40‐transformed mouse fibroblasts: Genetic and biochemical characterization
Authors: Benoni Désiré , Bradley W. E. C.
Published: February 2005

Mutants of SV40‐transformed mouse fibroblasts have been isolated that have greatly increased cell‐substratum adherence. The adherent phenotype (COL−) is recessive, and all mutants analyzed belong to one complementation group. No consistent qualitative differences between wild‐type and mutant cells were found with respect to the protein content of the substrate‐attached material (SAM), a cell surface fraction left after removal of cells from the substrate with a gentle Ca2+‐chelating agent. However, the mutants yielded 2.5‐10‐fold more SAM than the parental cell line, and the SAM deposited by mutants was able to mediate attachment of transformed cells to a much greater degree than was the SAM from the parental cell line. The mutation, which appears to control the generation of footpads, was shown to cosegregate with resistance to the drug 6‐thioguanine, which suggests X‐linkage.
A comparison of the platelet‐derived growth factor‐dependent tyrosine kinase activity in sparse and confluent fibroblasts
Authors: Kazlauskas Andrius , Dicorleto Paul E.
Published: February 2005

Confluent (density‐inhibited) human foreskin fibroblasts require a higher concentration of platelet‐derived growth factor (PDGF) to elicit a mitogenic response than do sparse (nondensity‐inhibited) fibroblasts. The PDGF receptor number and apparent affinity were similar in the two preparations of cells. The intrinsic kinase activity of the PDGF receptor from sparse and confluent fibroblasts was therefore examined in an attempt to explain the differential mitogenic response to PDGF. When membranes from sparse and confluent cells containing equal PDGF binding capacity were incubated with increasing concentrations of PDGF, the putative PDGF receptor (a 180‐kD component), was phosphorylated on its tyrosyl residues to a similar extent. The time course of tyrosine phosphorylation of the PDGF receptor from sparse and confluent cell membranes was also found to be similar. To determine whether the phosphorylation of the PDGF receptor from isolated membranes differed from the analogous phosphorylation in intact cells, sparse and confluent fibroblasts were metabolically labeled with [32P]H3PO4, stimulated with PDGF, solubilized, and the cell proteins were immunoprecipitated with a phosphotyrosine‐specific antibody. The extent of PDGF‐dependent tyrosine phosphorylation of the PDGF receptor from sparse vs. confluent fibroblasts was quite similar. The time course of the tyrosine dephosphorylation of the PDGF receptor was also similar in the two populations. Because comparable extents of PDGF‐induced tyrosine phosphorylation of the PDGF receptor were observed despite the differential PDGF‐induced mitogenic response of sparse and confluent fibroblasts, we tentatively conclude that (1) PDGF‐dependent tyrosine phosphorylation of the PDGF receptor is not tightly coupled to the propagation of the mitogenic signal and (2) density‐dependent inhibition of growth does not reflect any measurable change in the quantity of kinase activity of the PDGF receptor.
Electrophysiological differences between bone cell clones: Membrane potential responses to parathyroid hormone and correlation with the cAMP response
Authors: Ferrier Jack , Ward Angela
Published: February 2005

Electrophysiological measurements on three clonally derived bone cell populations showed a positive correlation between longer‐term hyperpolarizing membrane potential responses to parathyroid hormone (PTH) and an intracellular cAMP response to PTH. One clone (RCJ 1.20) had no sustained electrophysiological response and no cAMP response to PTH. Another clone (ROS 17/2.8) had both a sustained hyperpolarizing response and a cAMP response to PTH. The third clone (RCB 2.2) initially had both an electrophysiological response and a cAMP response to PTH, but both responses were lost after prolonged growth in culture. Application of dibutyryl cAMP to RCJ 1.20 and ROS 17/2.8 cells produced both transient and sustained hyperpolarizing responses. Application of isobutylmethylxanthine produced a sustained hyperpolarization. These results suggest that the hyperpolarizing response to PTH is related to a cAMP‐mediated increase in Ca2+ conductance, which leads to an increase in Ca2+‐activated K+ conductance. The pronounced membrane potential spikes and fluctuations that occur in some of the clonal lines were shown to be unrelated to the hyperpolarizing response to PTH. This was demonstrated by the lack of correlation between the occurrence of the spikes or fluctuations and the occurrence of the hyperpolarizing response to PTH in the various cell lines, by the lack of effect of PTH on the spikes and fluctuations, and by the lack of effect on the hyperpolarizing response to PTH of verapamil and quinine, both of which significantly reduce the spikes and fluctuations.
Tumor promoter enhances mitogenesis by PDGF with little effect on PDGF binding
Authors: Eide Brock L. , Krebs Edwin G. , Ross Russell , Pike Linda J. , Bowen‐Pope Daniel F.
Published: February 2005

Preincubation of Swiss 3T3 cells with the tumor promoter 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) at 37°C is observed to cause only a small (approximately 10%) decrease in maximal binding of 125l‐platelet‐derived growth factor (125I‐PDGF), and does not affect the affinity of 125I‐PDGF binding to these cells. Under the same conditions, the affinity of the epidermal growth factor receptor is greatly reduced, possibly resulting from phosphorylation by protein kinase C. TPA is also shown to have no effect on the kinetics of internalization or degradation of bound 125I‐PDGF. Although TPA has little or no effect on these properties of the PDGF receptor, it was found to act in a synergistic fashion with low, but not high, concentrations of PDGF to increase DNA synthesis by 3T3 cells. Since TPA has previously been shown to activate protein kinase C, these findings suggest that protein kinase C does not regulate the ligand‐binding properties of the PDGF receptor, and that the observed synergism between TPA and PDGF in stimulating mitogenesis reflects effects of TPA on other processes in the mitogenic pathway.
The role of erythropoietin in the production of principal erythrocyte proteins other than hemoglobin during terminal erythroid differentiation
Authors: Koury Mark J. , Bondurant Maurice C. , Mueller Thomas J.
Published: February 2005

Erythropoietin (EP) controls the terminal phase of differentiation in which proerythroblasts and their precursors, the colony forming units‐erythroid (CFU‐e), develop into erythrocytes. Biochemical studies of this hormone‐directed terminal differentiation have been hindered by the lack of a homogeneous population of erythroid cells at the developmental stages of CFU‐e and proerythroblasts that will synchronously differentiate in response to EP. Such a population of cells can be prepared from the spleens of mice with the acute erythroblastosis resulting from infection with anemia‐inducing Friend virus (FVA). Using these FVA‐infected erythroid cells, which were induced to differentiate with EP, four proteins other than hemoglobin that have key functions in mature erythrocytes were monitored during the 48‐hour period of terminal differentiation. Synthesis of spectrin and membrane band 3 proteins were determined by immunoprecipitation and SDS‐polyacrylamide gel electrophoresis; accumulation of the cytoskeletal protein band 4.1 was monitored by immunoblotting; carbonic anhydrase activity was measured electro‐metrically. Band 3 synthesis and band 4.1 accumulation could be detected only after exposure of the cells to EP. Spectrin synthesis was ongoing prior to culture with EP, but it did increase after exposure to the hormone. Carbonic anhydrase‐specific activity changed very little throughout the terminal differentiation process. These results reveal at least three patterns of production of principal erythrocyte proteins during EP‐mediated terminal differentiation of FVA‐infected erythroid cells. Depending on the specific protein examined, de novo synthesis can be induced by EP, an ongoing production can be enhanced by EP, or the production of a protein can be completed at a developmental stage prior to EP‐mediated differentiation in these cells.
Electrophoretic distribution and dissociation into subunits of lactate dehydrogenase derived from human myeloid leukemia cells before and after induction of differentiation
Authors: Pavelić Krešimir , Pekić Bosa
Published: February 2005

The electrophoretic distribution of lactate dehydrogenase (LDH) isoenzymes, Michaelis constant, reaction with substrate, and dissociation into subunits with guanidine hydrochloride was examined in undifferentiated and differentiated human myeloid leukemia cells. Differentiation was induced with 1/ μg/ml tunicamycin. Undifferentiated cells did not display phagocytic ability, and less than 5% of these cells had Fc receptors. After exposure to tunicamycin for 40 hr, 40% of these differentiated cells had Fc receptors, and 35% showed phagocytic activity after 160 hr. The majority of the LDH activity in the undifferentiated cells was found in fraction 3, and following differentiation almost a 50% reduction in LDH activity was observed in this fraction. In addition, LDH 3 isoenzyme levels were found to be greater in patients containing a high percentage of undifferentiated cells than in patients containing a high percentage of differentiated cells. Differentiated cells displayed LDH isoenzyme fraction pattern, Michaelis constant, and reaction with substrate similar to those found in the normal granulocytes. Differences in the dissociation of LDH into subunits with guanidine hydrochloride were found between undifferentiated and differentiated acute myeloid leukemia (AML) cells. Treatment with 0.75 M guanidine hydrochloride caused complete inactivation of LDH derived from normal differentiated cells, whereas similar treatment caused complete inactivation of LDH derived from AML or normal granulocytes. LDH isoenzymes derived from normal granulocytes and differentiated AML cells were also more sensitive to guanidine hydrochloride depression of fluorescence intensity. The sedimentation constant for single peak LDH at 5.5 M guanidine hydrochloride was calculated as 1.65 sec for differentiated and 1.70 sec for undifferentiated cells. The molecular weight of the polypeptide subunits for undifferentiated cells was 30,000 and for differentiated cells was 39,000. The apparent parallel between leukemic cells after induction of differentiation and normal granulocytes indicates that the leukemic cells retain their maturation potential when exposed to an inducer of differentiation.
Glutamine is responsible for stimulating glycolysis by L929 cells
Authors: Lanks Karl W.
Published: February 2005

L929 cells synthesize relatively little lactate when using glucose as their primary energy source, and neither glucose oxidation nor lactate production is highly sensitive to stimulation by insulin. Addition of glutamine to the system containing glucose and insulin markedly stimulates lactate production from glucose without inhibiting glucose oxidation. A mechanism in which reducing equivalents derived from glutamine oxidation are used to drive lactate production is discussed.
Posttranscriptional regulation of ornithine decarboxylase activity
Authors: Dircks Lori , Grens Ann , Slezynger Thelma C. , Scheffler Immo E.
Published: February 2005

We have used a Chinese hamster ovary cell line (DF3) that overproduces ornithine decarboxylase (ODC) to examine various parameters in the cell cycle‐dependent regulation of this enzyme. Under a variety of conditions, alterations in the activity of ODC were accompanied by parallel changes in the levels of the protein, as measured by immunologically cross‐reactive material (CRM). While putrescine has been known to suppress the induction of ODC, we have found that in DF3 cells 10−4M ornithine completely suppresses ODC activity. We also show that the levels of ODC mRNA are not modulated when the levels of ODC activity and CRM change drastically. The data can be interpreted in terms of models involving either an effect of putrescine on the translation of ODC mRNA, or on the activity of a relatively specific protease with ODC as its target.
The modulation of transcobalamin II (TC‐II) production by cyclic adenosine 3′,5′‐monophosphate in the murine macrophage cell line J774: Relationship to growth behavior
Authors: Melmed R. N. , Rachmilewitz B. , Schneider Aviva , Rachmilewitz M.
Published: February 2005

We undertook a study to define the role of cyclic AMP [cAMP] in modulating the secretion of transcobalamin II (TC‐II) in the mouse macrophage like cell line J774. J774 was observed to secrete large amounts of TC‐II, particularly in the presence of 8‐bromo cAMP or cholera toxin or when grown in medium supplemented with low concentrations of horse serum (1% or 5%) or in serum‐free medium. Variant cell lines derived from J774 and deficient either in adenylate cyclase (ac ‐ ) or cAMP‐dependent protein kinase (pk ‐ ) activity showed very low and intermediate levels of basal secretory activity of TC‐II, respectively, compared to J774. Maximum secretory activity of TC‐II was observed in J774 under conditions in which growth was poorest (in the presence of 8‐bromo‐cAMP or 1% or 5% horse serum‐supplemented medium or in serum‐free medium). Cells grown in serum‐free medium were found to have elevated basal adenylate cyclase activity and cAMP levels compared to those grown in medium supplemented with 20% horse serum. The data from this study demonstrate a negative correlation between growth activity and TC‐II secretion in the J774 cell line. The stimulatory effect of exogenous cAMP on TC‐II secretion by J774, the reduced secretory activity of the variant lines ac ‐ and pk ‐ and the observed increase in cell cAMP levels under conditions of serum starvation in which TC‐II secretion is considerably enhanced, suggest that cell cAMP is an important modulator of TC‐II secretion and growth behavior in the J774 cell line.
Evidence for a pool of non‐recycling transferrin receptors in peripheral sheep reticulocytes
Authors: Adam M. , Wu C. , Turbide C. , Larrick J. , Johnstone R. M.
Published: February 2005

Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that may exceed by an order of magnitude the surface binding capacity. Steady state uptake of transferrin at 37°C is generally less than 50% of the total transferrin binding capacity. During long‐term incubation of the reticulocytes, all transferrin binding ability is lost, the ability to internalize being lost most rapidly. The loss in ability to bind transferrin during long‐term incubation is independent of the number of surface transferrin binding sites, since removal of surface receptors with pronase does not affect the rate of loss of the internal pool of receptors during long‐term incubation. Moreover, after removing surface receptors with pronase, only a fraction of the original number of receptors is restored to the surface, despite the presence of a large pool of internal receptors. These data suggest that only a fraction of the internal pool of receptors is capable of recycling to the cell surface in sheep reticulocytes.
Alteration in cyclic adenosine 3′:5′‐monophosphate binding proteins during the phenotypic change of mouse macrophages transformed by a temperature‐sensitive mutant (tsA640) of SV40
Authors: Takayama Hisao , Tanigawa Takahiko , Tanaka Yoshinori , Onodera Kazukiyo
Published: February 2005

By using a photoaffinity ligand, cell extracts from transformed macrophages that were established by infection with temperature‐sensitive mutants (tsA640) of simian virus 40 (SV40) were examined for cyclic adenosine 3′:5′‐monophosphate (cAMP)‐binding proteins. At the nonpermissive temperature for SV40 large T antigen, 39.0°C, no significant cAMP‐binding proteins could be detected, such as primary mouse macrophages. At the permissive temperature of 33.0°C, cAMP‐binding proteins appeared later than SV40 T antigen expression and cellular DNA synthesis. The profile of cAMP‐binding proteins was similar to that of resting, but not proliferating, mouse clonal fibroblasts (BALB/c 3T3). These and previous results suggest that SV40 T antigen influences the expression of cAMP‐binding proteins in tsA640‐transformed macrophages; the large/small T antigen converts the profile of cAMP‐binding proteins from macrophage to fibroblastic cells.
Nuclear endogenous Ca2+‐dependent endodeoxyribonuclease in differentiating chick embryonic lens fibers
Authors: Muel Anne S. , Chaudun Elisabeth , Courtois Yves , Modak Sohan P. , Counis Marie F.
Published: February 2005

During terminal differentiation of lens epithelial cells into fiber cells, nuclei become pycnotic and DNA degradation occurs. We investigated the putative role in this process of an endogenous DNAase. After incubation of isolated nuclei of both cell types at 37°C, DNAase activity was revealed by DNA size analysis on 0.3–1% neutral and alkaline agarose, one‐ and two‐dimensional gels. This DNAase activity is more prominent in lens fiber nuclei than in epithelial nuclei at all the embryonic stages probably because of a preexisting higher concentration of divalent cations in the former. This activity is calcium or magnesium dependent in both types of nuclei.
Cellular basis for the species differences in sensitivity to cardiac glycosides (digitalis)
Authors: Gupta Radhey S. , Chopra Arvind , Stetsko Dawn K.
Published: February 2005

The relative toxicity of numerous cardiotonic steroids (viz. ouabain, digitoxin, digoxin, convallatoxin, SC4453, bufalin, gitaloxin, digoxigenin, actodigin, oleandrin, digitoxigenin, gitoxin, strophanthidin, gitoxigenin, lanatosides A, B and C, α‐ and β‐acetyl digoxin, α‐ and β‐methyl digoxin) and related compounds towards a number of independent cell lines established from human, monkey, mouse, Syrian hamster, and Chinese hamster have been determined. All cardiac glycosides and their genins, as well as the cardiotonic alkaloid cassaine, exhibited > 100‐fold higher toxicity towards cultured human and monkey cells in comparison to the cell lines of mouse, Syrian hamster, and Chinese hamster origins. These differences are species‐related as all cell lines (both normal as well as transformed) from any one species, as well as cells from the closely related species (e.g., man and monkey or mouse, Chinese hamster, and Syrian hamster), showed similar sensitivity towards these drugs. The failure to see any significant differences in cellular toxicity for a larger number of other compounds which either bear limited structural resemblance to cardiac glycosides (viz. estradiol 17‐β‐acetate, testosterone propionate, 21‐acetoxy pregnenolone, β‐estradiol, digitonin, tigogenin, and tomatine) or interact with the Na+/K+ ATPase in a different manner (viz. veratridine, sanguinarine nitrate, penicillic acid, vanadium pentoxide, harmaline‐HCI, 5,5′‐diphenyl hydantoin, quindonium bromide, and methyl quinolizinum bromide) provides strong evidence that the observed species‐related differences are highly specific for cardiotonic steroids. Studies on the binding of [3H]ouabain show that, in comparison to human and monkey cell lines, no significant binding of the drug is observed in cells derived from the resistant species (i.e., mouse and Chinese hamster). The Na+/K+ ATPase from cells of the resistant species is inhibited at much higher concentrations of ouabain and digitoxin in comparison to the enzyme from human cells, and a good correlation is observed between these concentrations and those reported for inhibition of the enzyme from isolated heart muscles of the same species. These results provide strong evidence that the species‐related differences in sensitivity to digitalis have a cellular basis and that the cultured cells from various mammalian species provide a useful model system for investigating the mechanism of action of cardiac glycosides.
Superoxide anion release by human endothelial cells: Synergism between a phorbol ester and a calcium ionophore
Authors: Matsubara Tsukasa , Ziff Morris
Published: February 2005

In order to study the signal transduction mechanism of human endothelial cells (EC), the regulation of superoxide anion (O2−) release in EC has been investigated using the calcium ionophore A23187 and phorbol myristate acetate (PMA), a potential activator of the Ca2+ activated, phospholipid‐dependent protein kinase, designated “protein kinase C.” PMA enhanced O2− release from EC, and this enhancement occurred regardless of the presence or absence of extracellular Ca2+. A similar increase was produced by A23187; omission of extracellular Ca2+ prevented this increase. Simultaneous stimulation with PMA and A23187 produced a large increase in O2− release at submaximal concentrations of these agents, which, when added separately, caused minimal effects. These findings indicate that the activation of protein kinase C and mobilization of Ca2+ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O2−.
Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells
Authors: Leitman Dale C. , Fiscus Ronald R. , Murad Ferid
Published: February 2005

The role of cyclic AMP on endothelial cell proliferation was investigated, since these cells can be exposed to high concentrations of physiological and pharmacological agents that alter cyclic AMP metabolism. Cloned bovine aortic endothelial cells were plated at 25,000 cells/35mm dish and grown for 5 days in the presence of phosphodiesterase (PDE) inhibitors, forskolin, or cyclic AMP analogs. The PDE inhibitors dipyridamole, ZK 62 711, isobutylmethylxanthine (IBMX) and theophylline inhibited cell growth in a concentration‐dependent manner. Dipyridamole produced a 30% and a 50% inhibition at 5μM and 12.5μM, while higher concentrations were cytotoxic. At its therapeutic plasma concentration range (50‐100μM) theophylline inhibited cell proliferation by 15–25%, while IBMX and the highly specific cyclic AMP phosphodiesterase inhibitor, ZK 62 711 inhibited growth by 60–80% and 40–50%, respectively. Forskolin (5μM) increased cyclic AMP levels and cyclic AMP‐kinase activity ratios by 2.5‐fold and 2‐fold. In the absence of PDE inhibitors forskolin produced a 20% growth inhibition at 0.5μM and a 60% inhibition at 10μM. The forskolin dose‐response curve was not altered by theophylline, but was shifted to the left by approximately 10‐fold with dipyridamole and ZK 62 711 and 5‐fold with IBMX. Forskolin (5μM), by itself produced a 1.8‐fold increase in cyclic AMP. In the presence of 5μM theophylline, dipyridamole, IBMX, and ZK 62 711, cyclic AMP was increased by forskolin 2.0, 2.6, 3.5, and 6.6‐fold, respectively. 8‐Bromo cyclic AMP and dibutyryl cyclic AMP produced a 55% and 60% growth inhibition at 100μM. The cyclic GMP analogs were less effective inhibitors of growth (15–30%). Our results demonstrate that cyclic AMP analogs and pharmacological agents that elevate intracellular cyclic AMP levels inhibit cell growth and suggest that cyclic AMP may be an important endogenous regulator of endothelial cell proliferation.
Expression of phenotypic traits following modulation of colchicine resistance in J774.2 cells
Authors: Lothstein Leonard , Horwitz Susan Band
Published: February 2005

Development of resistance to colchicine in the mouse macrophage‐like cell line J774.2 coincides with the expression of a variety of phenotypic traits. A cloned subline (J7/CLC‐20), maintained in 20 μM colchicine, exhibits reduced steady‐state association with drug, increased presence of a 140,000–145,000 dalton (140–145 kD) phosphoglycoprotein associated with the plasma membrane, double minute chromosomes and cross‐resistance to other drugs. While similar phenotypic traits are observed in J774.2 cells resistant to taxol and vinblastine, differences in the electrophoretic mobilities of the resistance‐specific glycoproteins in each of the three sublines suggest that multi‐drug resistant sublines exhibit specificity for individual drugs. In an attempt to elucidate the relationships between the phenotypic traits associated with colchicine resistance, the degree of colchicine resistance in J7/CLC‐20 cells was modulated and the levels of expression of the phenotypic traits were quantitated. In the absence of colchicine in the growth medium, J7/CLC‐20 cells reverted to drug sensitivity within 35 days. A decrease in the level of resistance coincided with coordinate changes in both the quantity of the resistance‐specific glycoprotein and the average number of double minute chromosomes. We propose that the emergence and disappearance of the resistance‐specific glycoprotein and double minute chromosomes may be closely linked. However, J7/CLC‐20 cells which had regained their drug sensitivity after growth in drug‐free medium maintained a reduced level of steady‐state drug association. The persistence of reduced drug association in cells that have reverted to a drug‐sensitive state suggests that this phenomenon, although related to colchicine resistance, need not be the primary or only mechanism of drug resistance.
Accumulation of cells with 4N DNA content at nonpermissive temperature in rat embryo diploid cells transformed by tsA mutant of simian virus 40
Authors: Okuda Atsuyuki , Tamura Hideaki , Shimura Hideo , Kimura Genki
Published: February 2005

Primary rat embryo cells were transformed by a tsA mutant (tsA640) of simian virus 40 (SV40). Proliferation of all four independent diploid transformants was suppressed at a nonpermissive temperature (40.3°C), being accompanied by a marked increase in the fraction of cells with a 4N DNA content (a 4N peak in the flow cytofluorogram). However, in this case, the fraction of cells with a 2N DNA content (a 2N peak in the flow cytofluorogram) was preserved. Both effects (suppression of proliferation and increase in the 4N peak) diminished when transformed cells were superinfected with wild‐type SV40. The increased 4N peak was preserved, albeit not completely, for at least 24 hours, when cells were further incubated in the presence of hydroxyurea at the nonpermissive temperature. On the other hand, the preserved 2N peak all but disappeared within 24 hours, when cells were further incubated in the presence of colcemid at the nonpermissive temperature. These results suggest that the thermolabile large T antigen of SV40 directly or indirectly induces an accumulation of cells with a 4N DNA content, at the nonpermissive temperature, by prolonging the G2 (and/or late S) period.
Characterization of water in unfertilized and fertilized sea urchin eggs
Authors: Merta Philip J. , Fullerton Gary D. , Cameron Ivan L.
Published: February 2005

The water in unfertilized and fertilized sea urchin eggs was characterized with a proton nuclear magnetic resonance (NMR) titration method assuming fast proton diffusion (FPD) between water compartments. This method involves stepwise dehydration with sequential T1 relaxation time and water content determinations. The results analyzed by the FPD model give evidence of intracellular water compartments with three different correlation times: 6 × 10−12 sec (bulk water), 1 × 10−10 sec (structured water) and about 2 × 10−9 sec (bound water). Fertilization is accompanied by a substantial increase in bulk water (from 111 to 414 g H2O per 100 g dry mass) and by a decrease in the water of hydration (from 128 g to 56 g per 100 g dry mass). This study shows that 54% of the water in the unfertilized sea urchin egg has motional properties different from bulk water and that this percentage decreases dramatically shortly after fertilization. Most of the change in T1 relaxation rate observed at fertilization can be accounted for by uptake of bulk water associated with elevation of the fertilization membrane.
Temporal order of replication of genes responsible for hypoxanthine phosphoribosyl transferase and Na+/K+ ATPase in chemically transformed human fibroblasts
Authors: Tsutsui Takeki , Suzuki Nobuko , Elmore Eugene , Maizumi Heiji
Published: February 2005

The cytotoxic and mutagenic effects of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of a chemically induced, transformed line (Hut‐11A cells) derived from diploid human skin fibroblasts were examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2‐h periods during the S phase with 5‐bromodeoxyuridine (BrdU) followed by irradiation with near‐ultraviolet (UV). The BrdU‐plus‐irradiation treatment was cytotoxic and mutagenic, while treatment with BrdU alone or irradiation alone was neither cytotoxic nor mutagenic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 h of S phase, whereas scare lethality was observed in late S phase. The BrdU‐plus‐irradiation treatment induced ouabain‐ and 6‐thioguanine‐resistant mutants, while BrdU alone or irradiation alone was not mutagenic. Ouabain‐resistant mutants were induced during early S phase by the BrdU‐plus‐irradiation treatment. 6‐Thioguanine‐resistant mutants, however, were induced during middle to late S phase. These results suggest that a certain region or regions in the DNA of Hut‐11A cells, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by BrdU treatment plus near‐UV irradiation for cell survival and that gene(s) responsible for Na+/K+ ATPase is replicated during early S phase and gene(s) for hypoxanthine phosphoribosyl transferase is replicated during middle to late S phase.
Regulation of cell attachment and cell number by fibronectin and laminin
Authors: Terranova Victor P. , Aumailley Monique , Sultan Leslie H. , Martin George R. , Kleinman Hynda K.
Published: February 2005

We have examined the effect of laminin and fibronectin on the attachment and growth on type IV collagen of a line of mouse epithelial cells and a strain of adult human fibroblasts. Laminin stimulated attachment of the epidermal cells and fibronectin stimulated fibroblast attachment. At high concentrations (100 μg/ml), the attachment proteins altered the growth of cells in culture. The epidermal cells grew better in media containing fibronectin‐free serum supplemented with laminin. Fibroblasts, on the other hand, grew best in media containing serum supplemented with fibronectin. These data suggest that laminin promotes epithelial cell growth whereas fibronectin promotes fibroblast growth. This observation was confirmed when these cells were cocultured in the presence of the attachment proteins or of their respective antibodies. The mouse epidermal cells grew best when laminin was added to cocultures of fibroblasts and epithelial cells. Fibroblasts grew best in the presence of antibody to laminin and poorly in the presence of antibody to fibronectin. Thus, fibronectin and laminin may participate in the regulation of cell populations in vivo and may be involved in epithelial‐mesenchymal interactions.
The role of intracellular pH in ligand internalization
Authors: Hwang Jaulang , Pouyssegur J. , Willingham M. C. , Pastan I.
Published: February 2005

Internalization of EGF and transferrin measured as the rate of uptake of 125l‐labeled ligands was compared in the cell line CCL39 and a mutant derivative, PS120, lacking the Na+/H+ antiport system. No significant alteration was detected between the two cell lines. In contrast, pretreatment of the mutant cells PS‐120 with 20 mM NH4Cl for 30 min to decrease persistently intracellular pH resulted in an increase in 125I‐EGF and 125I‐transferrin uptake by 60% and 25%, respectively. However, similar NH4Cl pretreatment of the parental cell line, CCL‐39, which only affected intracellular pH very transiently did not cause an increase of ligand uptake. The binding of 125I‐EGF to CCL‐39 and PS‐120 cells with or without NH4Cl pretreatment showed that NH4Cl pretreatment did not affect EGF binding in either CCL‐39 or PS‐120 cells. Since cells regulate intracellular pH by ion transport systems, we also examined the role of Na+, K+‐ATPase. Ouabain, an inhibitor of Na+, K+‐ATPases, showed no effect on 125I‐EGF uptake in either of the cell types with or without NH4Cl pretreatment. Taken together, these results suggest that the plasma membrane‐bound Na+/H+ antiport, a major pHi‐regulating system in vertebrates, indirectly plays a role in ligand internalization through regulation of intracellular pH.
Control of type IV collagen production in rat mammary epithelial and myoepithelial‐like cells
Authors: Warburton Michael J. , Kimbell Rosemary , Rudland Philip S. , Ferns Sharon A. , Barraclough Roger
Published: February 2005

A rat mammary myoepithelial‐like cell line (Rama 401) produces 3.5 times more type IV collagen than a mammary epithelial cell line (Rama 25), as measured by the formation of protein hydroxyproline. However, using quantitative “dot” hybridization techniques, the level of poly (A)‐ containing mRNA hybridizing to a type IV collagen cDNA probe is only 50% higher in Rama 401 cells than in Rama 25 cells. The total amount of hydroxyproline synthesized per cell by the two cell lines is similar. However, in the Rama 25 cells approximately 70% of the hydroxyproline is found as free hydroxyproline against 13% for Rama 401 cells. When Rama 25 cells are grown on collagen gels, they accumulate 2.5‐fold more type IV collagen. However, type IV collagen mRNA levels are only 30% higher in Rama 25 cells grown on collagen. The total amount of hydroxyproline synthesized is the same as cells grown on plastic, whereas the extent of collagen degradation is reduced from 70% to 30% in cells grown on collagen gels. No degradation of type IV collagen can be detected in the culture medium of Rama 25 cells. These results indicate that the increased accumulation of type IV collagen in Rama 401 cells is not due to increased synthesis but to a decreased rate of intracellular degradation, and that for Rama 25 cells, the extracellular matrix modulates type IV collagen production by regulating the rate of intracellular collagen degradation.
Glucocorticoid‐induced heat resistance in mammalian cells
Authors: Fisher George A. , Anderson Robin L. , Hahn George M.
Published: February 2005

Chinese hamster ovary cells were incubated for 24 h in a variety of steroid hormones (testosterone, progesterone, hydrocortisone, dexamethasone, and ecdysterone) to test their effect on the subsequent heat resistance of the cells. Only the glucocorticoids, hydrocortisone and dexamethasone, consistently induced heat resistance. Heat resistance induced by hydrocortisone at 10−6M developed after a lag of 2–3 h and was maximal by 20 h. Resistance was expressed in both asynchronous and plateau phase cells and was maintained for several days in medium without added hormone. Incubation of cells with hydrocortisone and a 100‐fold excess of progesterone (a glucocorticoid antagonist) partially inhibited the development of resistance. Prior exposure to hydrocortisone did not inhibit the subsequent development of heat induced thermotolerance. However, cells made thermotolerant by prior heat shock did not display further heat resistance with hydrocortisone treatment. There was no evidence for the induction of heat shock proteins (HSP) by these steroid hormones although the 28 kDHSP was further enhanced by combined heat and hydrocortisone. Our results indicate that heat resistance in mammalian cells may be induced by physiological concentrations of glucocorticoids and that the characteristics of this resistance are consistent with a receptor mediated event.
Mitogenic activity of hydroxyapatite: Requirement for somatomedin C
Authors: Cheung H. S. , Van Wyk J. J. , Russell W. E. , McCarty D. J.
Published: February 2005

Synovial hyperplasia is a feature of the chronic synovitis associated with basic calcium phosphate crystals [hydroxyapatite (HA), octacalcium phosphate, tri‐calcium phosphate] and calcium pyrophosphate. Each of these crystals stimulated mitosis of cultured human skin fibroblasts or canine synovial fibroblasts in a concentration‐dependent fashion. We examined the effect of pure somatomedin C (Sm‐C) on HA crystal induced mitogenesis. Confluent cultures of human fibroblasts were rendered quiescent by incubation in the presence of 1% platelet‐poor‐Sm‐C free plasma (PPSCFP) for 24 hours. HA crystals stimulated thymidine incorporation 2.3‐fold over control value. Addition of Sm‐C significantly augmented the effect of HA crystals (P < 0.01). Nearly identical effects were observed in the presence of 100 μg/ml HA crystals or 15 ng/ml PDGF. Monoclonal antibodies against Sm‐C had little effect on the basal 3H thymidine uptake by control cells incubated in 1% PPSCFP but blocked over 50% of the HA crystal of PDGF‐induced 3H thymidine incorporation both in the presence or absence of Sm‐C. The incomplete blocking suggested either the presence of other “progression” factors, such as insulin‐like growth factor II in the conditioned media or the possibility that HA or PDGF in high enough dosage enabled cells to escape their dependence on Sm‐C for DNA synthesis.
Prostaglandin E2 stimulates DNA synthesis by a cyclic AMP‐independent pathway in osteoblastic clone MC3T3‐E1 cells
Authors: Hakeda Yoshiyuki , Yoshino Takahiko , Natakani Yoshinori , Kurihara Noriyoshi , Maeda Norihiko , Kumegawa Masayoshi
Published: February 2005

The effect of prostaglandin E2 (PGE2) on osteoblastic cell proliferation was investigated using osteoblastic clone MC3T3‐E1 cells cultured in serum‐free medium. PGE2 at 2 μg/ml increased the number of the cells by 2 days after its addition. PGE2 raised the level of DNA synthesis in a dose‐related fashion after a constant lag time, the maximal effect being at 2–10 μg/ml and the level about fourfold over that of the control at 36 hr after its addition. However, at low doses (below 0.2 μg/ml), PGE2 rather depressed DNA synthesis. Isobutyl methylxanthine counteracted the stimulation of DNA synthesis by PGE2, and forskolin depressed the synthesis, which was inversely correlated with increasing intracellular cAMP content. These results indicate that an increase in cAMP content inhibits DNA synthesis. In addition, 2′,5′‐dideoxyadenosine did not negate the stimulatory effect of PGE2 on DNA synthesis, suggesting that PGE2 increases DNA synthesis, probably via a pathway different from the adenylate cyclase/cAMP system. Moreover, at a high dose, PGE2 stimulated both the production and degradation of cAMP; the elevationof cAMP content was rapidly depressed by the stimulated degradation system. Consequently, thestimulatory effect of PGE2 on DNA synthesis would be released from the inhibition by cAMP, resulting in an increase in DNA synthesis. Taken together with data from our previous reports, these results indicate that PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.