bluuebunny/crossref_metadata_2025 huggingface dataset:
Renewed DNA synthesis in senescent WI‐38 cells by expression of an inducible chimeric c‐fos construct
Authors: Phillips Paul D. , Pignolo Robert J. , Nishikura Kazuko , Cristofalo Vincent J.
Published: February 2005

As normal human cells approach the end of their proliferative lifespan in vitro they lose responsiveness to a variety of growth factors, to which they respond with DNA replication when they are young. Recently it has been reported that the protooncogene c‐fos is not expressed in senescent cells (Seshadri and Campisi, 1990). In this study we have found that both c‐jun and jun B, partners of c‐fos in heterodimeric transactivating complexes, are equivalently expressed in young and senescent cells at both early (1–6 hr) and late (12 or 16 hr) time points following serum stimulation of quiescent cells. We have also investigated the effect of the enforced expression of c‐fos in senescent WI‐38 cells using an inducible construct carrying the murine c‐fos gene under the control of the sheep metallothionein promoter. We have found that the transient transfection and subsequent activation of the conditional promoter with Zn++ stimulated DNA synthesis in a significant fraction of senescent cells which had completed 90%–95% of their proliferative lifespan. However, populations which had completed 100% of their proliferative life span and nondividing cultures which had been selected with BrdU did not respond to the expression of the c‐fos gene. These results demonstrate that one of the primary events associated with senescence in human cells is the suppression of c‐fos gene expression, but additional phenotypic changes must also occur in order to explain the ultimate loss of proliferative responsiveness of these cells. © 1992 Wiley‐Liss, Inc.
Control of alkaline phosphatase activity in C3H10T1/2 cells: Role of retinoic acid and cell density
Authors: Reese David H. , Larsen Rachel A. , Hornicek Francis J.
Published: February 2005

The enzyme alkaline phosphatase (AP) has been shown to be lost or inappropriately expressed during carcinogenesis in some tissues. Because retinoic acid (RA) appears to play a role in the normal regulation of the enzyme (RA up‐regulates AP in a variety of cell types) we have suggested that altered AP expression in some cancers may be caused by a defect in the ability of the cells to respond normally to retinoid. We have begun to use the chemically transformable mouse embryo fibroblast cell, C3H10T1/2, to investigate this possibility. In this initial study we characterized AP regulation in normal C3H10T1/2 cells and show that: (1) 10−7 M RA increases AP activity within 3–4 h in serum‐free medium; (2) serum inhibits short‐term induction (0–8 h) in a concentration‐dependent manner (10% serum causes complete inhibition); (3) during long‐term RA exposure (24 h and 48 h), induction can be detected in serum‐containing medium; (4) AP induction is dose related at RA concentrations from 10−10 M to 10−6 M in serum‐free medium; (5) 10−5 M RA is ineffective at inducing AP in serum‐free medium during 8 h but is the most effective concentration in serum‐containing medium during 24 h and 48 h exposures; (6) AP inducibility by RA requires near‐confluent cell densities; and (7) when cultures become confluent, cells become constitutive for AP and no longer require RA for enzyme expression. The effects of serum and cell density on AP inducibility by RA and implications of the RA up‐regulation of AP for teratogenesis are discussed. © 1992 Wiley‐Liss, Inc.
The inward rectifier potassium conductance in rat basophilic leukemia cells
Authors: Piguet Pascale , North R. A.
Published: February 2005

Whole‐cell recordings were made from rat basophilic leukemia cells. The properties of the cells in the potential range negative to −20 mV could be accounted for by two potassium conductances, a leakage conductance (0.54 ± 0.01 nS, mean ± s.e.m., n − 6) and an inward rectifier conductance. The inward rectifier conductance activated at a potential close to Ek and had a maximal value of 2.7 ± 0.22 nS (n = 6 ). Cesium (2 mM, n = 6) decreased the inward current at every potential. Strontium (10 mM, n = 7) and rubidium (2 mM, n = 3) had a similar effect, but they also increased the slope conductance between −63 and − 103 mV. Barium (1–100 pM) blocked selectively the inward rectifying current without affecting the leakage current; the effect was use‐, voltage‐, and temperature‐dependent. TEA decreased the current; the concentration giving 50% inhibition was 37.5 mM. The current was unaffected by phorbol esters as well as several hormones and transmitters that werg tested. © 1992 Wiley‐Liss, Inc.
Relationship between thermal tolerance and protein degradation in temperature‐sensitive mouse cells
Authors: Li Jian Jian , Dewey William C.
Published: February 2005

The induction of thermotolerance was studied in a temperature sensitive mouse cell line, ts85, and results were compared with those for the wild‐type FM3A cells. At the nonpermissive temperature of 39°C, ts85 cells are defective in the degradation of short‐lived abnormal proteins, apparently because of loss of activity of a ubiquitin‐activating enzyme. The failure of the ts85 cells to develop thermotolerance to 41–43°C after incubation at the nonpermissive temperature of 39°C correlated with the failure of the cells to degrade short‐lived abnormal proteins at 39°C. However, the failure of the ts85 cells to develop thermotolerance to 43°C during incubation at 33°C after either arsenite treatment or heating at 45.5°C for 6 or 10 min did not correlate with protein degradation rates. Although the rate of degrading abnormal protein was reduced after heating at 45.5°C for 10 min, the rates were normal after arsenite treatment or heating at 45.5°C for 6 min. In addition, when protein synthesis was inhibited with cyclohexmide both during incubation at 33°C or 39°C and during heating at 41–43°C, resistance to heating was observed, but protein degradation rates at 39°C or 43°C were not altered by the cycloheximide treatment. Therefore, there is apparently no consistent relationship between rates of degrading abnormal proteins and the ability of cells to develop thermotolerance and resistance to heating in the presence of cycloheximide. © 1992 Wiley‐Liss, Inc.
Induction of differentiation and c‐jun expression in human leukemic cells by okadaic acid, an inhibitor of protein phosphatases
Authors: Adunyah Samuel E. , Unlap Tino M. , Franklin Christopher C. , Kraft Andrew S.
Published: February 2005

Okadaic acid, a protein phosphatase inhibitor, is a strong tumor promoter which activates protein phosphorylation. Because another activator of protein phosphorylation, phorbol esters, stimulates hematopoietic differentiation, we sought to determine whether okadaic acid could also induce the differentiation of the human leukemic cell line U937. Differentiation was assessed by measuring changes in the following: mRNA levels, cell growth, morphology, cell surface markers, and the ability to induce superoxide. We found that okadaic acid treatment of U937 cells induces immediate increases in total cellular levels of both c‐junand c‐fos mRNAs. Nuclear run‐on experiments demonstrate that initial increases are secondary to increases in transcription, whereas latter changes may be secondary to mRNA stabilization. Like phorbol esters, okadaic acid treatment also activates AP‐1 enhancer activity and induces the phosphorylation of c‐Jun protein. Approximately 6–12 hours after treatment with okadaic acid, mRNA levels of c‐myc, p34cdc2, and p58GTA, two cell cycle regulated protein kinases, decrease. Okadaic acid inhibits the growth of U937 cells, induces changes in nuclear morphology, stimulates increases in Mac‐1 and Leu 11 surface antigens, and induces these cells to produce superoxide. These changes, taken together, suggest that U937 cells have been induced by okadaic acid to differentiate towards a more mature cell type. © 1992 Wiley‐Liss, Inc.
Stimulation of endothelial cell proliferation by vanadate is specific for microvascular endothelial cells
Authors: Maher Pamela A.
Published: February 2005

Micromolar concentrations of sodium orthovanadate stimulated the proliferation of bovine capillary endothelial cells, but not bovine aortic endothelial cells. Vanadate was equally potent at inducing protein tyrosine phosphorylation and changes in morphology in both types of cells. However, vanadate treatment lead to an inhibition of protein tyrosine kinase activity in the aortic endothelial cells, but not the capillary endothelial cells. In capillary endothelial cells, the effect of vanadate was additive with basic FGF (bFGF) at low concentrations of bFGF. There was no interaction between bFGF and vanadate in aortic endothelial cells. TGF‐β, which inhibits the induction of endothelial cell proliferation by bFGF, appeared to shift the dose response curve to vanadate in capillary endothelial cells, increasing the proliferative effect of vanadate at low vanadate concentrations, but decreasing the proliferative effect at higher vanadate concentrations. © 1992 Wiley‐Liss, Inc.
Adenosine receptors, cyclic AMP accumulation, and DNA‐synthesis in aortic smooth muscle cell cultures of adult and neonatal rats
Authors: Querol‐Ferrer Valérie , Hultgardh‐Nilsson Anna , Ringertz Nils R. , Nilsson Jan , Jonzon Bror
Published: February 2005

The effects of two adenosine analogs on cyclic AMP (cAMP) accumulation and DNA synthesis were studied in cultured smooth muscle cells (SMCs) isolated from adult and neonatal rat arteries. N‐ethylcarboxamido adenosine (NECA) dosedependently increased intracellular cAMP levels and appeared to be more potent in adult than in neonatal SMCs. R‐phenylisopropyl adenosine (R‐PIA), in nanomolar concentrations, counteracted the increase in cAMP evoked by 10 μM forskolin in adult but not in neonatal SMCs, indicating that the enhanced “A2” response seen in adult SMCs was not due to a lack of “A1” receptors in these cultures. Binding experiments performed using the adenosine antagonist XAC did not reveal any differences in the number or affinity of the adenosine receptors between neonatal and adult SMCs. This indicates effects presumably on the G‐protein level. A high capacity to spontaneously synthesize DNA and a weak response to platelet‐derived growth factor (PDGF) were seen in the neonatal SMCs. Furthermore, NECA had no effect on PDGF‐induced DNA synthesis in these cells. In contrast, adelt SMCs presented a low rate of spontaneous DNA synthesis and a marked proliferative response to PDGF, which was inhibited by NECA. This inhibition paralleled the increase in cAMP elicited by NECA. Our findings suggest that neonatal and adult SMCs differ both in their response to growth factors and growth inhibitors. © 1992 Wiley‐Liss, Inc.
Quick and accurate method to convert BCECF fluorescence to pHi: Calibration in three different types of cell preparations
Authors: James‐Kracke Marilyn R.
Published: February 2005

A rapid, easy, and accurate method for converting the fluorescence of BCECF to pH, as an alternative to the nigericin method, is described. The ratio of the fluorescence intensities for BCECF can be converted to pH between 4 and 9 by a formula similar to the one used to calculate [Ca2+]i from the fluorescence of fura2. The formula is inverted because H+ binding to BCECF causes a decrease in fluorescence, whereas Ca2+ binding to fura2 causes an increase in fluorescence. The ratio of the fluorescence intensities is a sigmoidal function of the [H+] between pH 4 and 9 with an essentially linear mid region from pH 6 to 8. This calibration procedure in cells is similar to the popular method for fura2 where ionomycin, Ca2+, and an alkaline EGTA solution are added in succession to change the intracellular pCa from 4 to 9. For BCECF in cells, a protonophore, FCCP or CCCP, is added and the cells are titrated with acid to an intracellular pH of 4 and then back to pH 9 with base by observing the gradual change in fluorescence as it asymptotically reaches its limiting minimum and maximum values. This method does not require changing the medium to one with high KCl to depolarize the membrane potential nor does the proton concentration need to be equilibrated across the plasma membrane. The technique can be used to calibrate BCECF in sheets of cells, as well as suspensions of cells over a wide range of pH sensitivities. © 1992 Wiley‐Liss, Inc.
Biochemical events accompanying macrophage activation and the inhibition of colony‐stimulating factor‐1‐induced macrophage proliferation by tumor necrosis factor‐α, interferon‐γ, and lipopolysaccharide
Authors: Vairo Gino , Royston A. Keith , Hamilton John A.
Published: February 2005

Agents that can arrest cellular proliferation are now providing insights into mechanisms of growth factor action and how this action may be controlled. It is shown here that the macrophage activating agents tumor necrosis factor‐α (TNFα), interferon‐γ (IFNγ), and lipopolysaccharide (LPS) can maximally inhibit colony stimulating factor‐1 (CSF‐1)‐induced, murine bone marrow‐derived macroohage (BMM) DNA synthesis even when added 8–12 h after the growth factor, a period coinciding with the G1/S‐phase border of the BMM cell cycle. This inhibition was independent of autocrine PGE2 production or increased cAMP levels. In order to compare the mode of action of these agents, their effects on a number of other BMM responses in the absence or presence of CSF‐1 were examined. All three agents stimulated BMM protein synthesis; TNFα and LPS, but not IFNγ, stimulated BMM Na+/H+ exchange and Na+, K+‐ATPase activities, as well as c‐fos mRNA levels. IFNγ did not inhibit the CSF‐1‐induced Na+, K+‐ATPase activity. TNFα and LPS inhibited both CSF‐1‐stimulated urokinase‐type plasminogen activator (u‐PA) mRNA levels and u‐PA activity in BMM, whereas IFNγ lowered only the u‐PA activity. In contrast, LPS and IFNγ, but not TNFα, inhibited CSF‐1‐induced BMM c‐myc mRNA levels, the lack of effect of TNFα dissociating the inhibition of DNA synthesis and decreased c‐myc mRNA expression for this cytokine. These results indicate that certain biochemical responses are common to both growth factors and inhibitors of BMM DNA synthesis and that TNFα, IFNγ, and LPS, even though they all have a common action in suppressing DNA synthesis, activate multiple signaling pathways in BMM, only some of which overlap or converge. © 1992 Wiley‐Liss, Inc.
Demonstration of an insulin‐insensitive storage pool of glucose transporters in rat hepatocytes and HepG2 cells
Authors: Hah Jongsik , Jo Inho , Chakrabarti R. , Jung Chan Y.
Published: February 2005

The subcellular distribution of glucose transporters in rat hepatocytes and HepG2 cells was studied in the absence and in the presence of insulin. Glucose transporters were quantitated by measuring glucose‐sensitive cytochalasin B binding and by protein immunoblotting using isoform‐specific antibodies. Plasma membrane contamination into subcellular fractions was assessed by measuring distribution of 5′‐nucleotidase and cell surface carbohydrate label. In hepatocytes, GLUT‐2 occurred in a low‐density microsomal (LDM) fraction at a significant concentration, and as much as 15% of cellular GLUT‐2 was found intracellularly that cannot be accounted for by plasma membrane contamination. In HepG2 cells which express GLUT‐1 and GLUT‐2, the two isoforms showed distinct subcellular distribution patterns: GLUT‐2 was highly concentrated in LDM while very little GLUT‐1 was found in this fraction, indicating that a large portion of GLUT‐2 occurs in intracellular organelles. Insulin treatment did not change the subcellular distribution patterns of glucose transporters in both cell types. Our results suggest that rat hepatocytes and HepG2 cells possess an intracellular storage pool for GLUT‐2, but lack the insulin‐responsive glucose transporter translocation mechanism. © 1992 Wiley‐Liss, Inc.
Rapid inverse changes in α1B‐and β2‐adrenergic receptors and gene transcripts in acutely isolated rat liver cells
Authors: Ishac Edward J. N. , Lazar‐Wesley Eliane , Kunos George
Published: February 2005

In vitro incubation of hepatocytes acutely isolated from adult male rats leads to a rapid conversion of the adrenergic activation of glycogenolysis from an α1‐receptor (α1AR) to a β2‐receptor (β2AR) mediated response within 4 h. In order to understand the underlying mechanism, we examined time‐dependent changes in α1‐ and β2‐adrenergic activation of glycogenolysis and second messenger systems, the cellular density and affinity of α1AR and β2AR, and the steady state levels of α1BAR and β2AR mRNAs. Incubation of hepatocytes for 4 h resulted in a decrease in phosphorylase activation and inositol 1,4,5 trisphosphate accumulation in response to phenylephrine, a 40% decrease in α1AR density, and a 70% decrease in α1BAR mRNA levels. Incubation of hepatocytes for 4 h also resulted in the emergence of a phosphorylase response to isoproterenol, an increase in isoproterenol‐induced but not in glucagon‐ or forskolin‐induced cAMP accumulation, no significant change in β2AR density, and a twofold increase in β2AR mRNA levels. Exposure of cells to cycloheximide, 2 μM throughout the 4 h incubation, prevented the emergence of the phosphorylase response to isoproterenol and reduced β2AR densities, while the decrease in α1AR density was not affected and the decrease in phosphorylase activation by phenylephrine was attenuated. The results indicate that dissociation of rat liver cells triggers a rapidly developing decrease in α1BAR mRNA and increase in β2AR mRNA levels and corresponding inverse changes in the synthesis of α1BAR and β2AR which account, at least in part, for the rapid conversion from α1‐ to β2‐adrenergic glycogenolysis. © 1992 Wiley‐Liss, Inc.
Reduced mRNA levels for the multidrug‐resistance genes in cAMP‐dependent protein kinase mutant cell lines
Authors: Chin Khew‐Voon , Chauhan Shyam S. , Abraham Irene , Sampson Kathleen E. , Krolczyk Arkadiusz J. , Wong Margaret , Schimmer Bernard , Pastan Ira , Gottesman Michael M.
Published: February 2005

We have previously shown that in Chinese hamster ovary (CHO) cells, a mutant cell line with a defective regulatory subunit (RI) for the cAMP‐dependent protein kinase (Abraham et al: Mol. Cell. Biol., 7:3098–3106, 1987), and a transfectant cell line expressing the same mutant kinase, showed increased sensitivity to a number of drugs that are known to be substrates for the multidrug transporter (P‐glycoprotein). In the current study we have investigated the mechanism by which cAMP‐dependent protein kinase controls drug resistance. We report here that the sensitivity of the kinase defective CHO cell lines to multiple drugs results from decreased RNA levels for the multidrug‐resistance gene. Similar results were obtained with mouse Y1 adrenal cells. Wild‐type Y1 cells had high levels of P‐glycoprotein due to expression of both the mdr 1b and mdr2 genes, whereas the cAMP‐dependent protein kinase mutant Kin 8 cells had decreased RNA levels for these genes. A Kin 8 transfectant with restored cAMP‐dependent protein kinase activity recovered mdr expression, indicating a cause and effect relationship between the protein kinase mutations and mdr expression. No changes in nuclear run‐off assays could be detected, suggesting a non‐transcriptional mechanism of regulation. Wild‐type Y1 cells are more drug sensitive despite having higher levels of P‐glycoprotein than the mutant cells. This paradoxical result may be explained by the higher rate of synthesis of steroids by the wild‐type Y1 cells, which appear to be inhibitors of P‐glycoprotein transport activity. © 1992 Wiley‐Liss, Inc.
Basolateral distribution of fibronectin matrix assembly sites on vascular endothelial monolayers is regulated by substratum fibronectin
Authors: Kowalczyk Andrew P. , McKeown‐Longo Paula J.
Published: February 2005

Endothelial cells exhibit binding sites for the amino terminus of fibronectin that participate in subendothelial fibronectin matrix assembly. These binding sites, termed matrix assembly sites, are localized on the basolateral surface of confluent endothelial monolayers (Kowalczyk et al. Blood, 75:2335, 1990). The present study investigates the role of cell—cell and cell—substratum interactions in the localization of matrix assembly sites to the basal surface of endothelial cells. Cells were cultured in Transwell culture inserts and matrix assembly sites were detected by binding assays using an iodinated 70 Kd amino‐terminal fibronectin fragment. Integrity of intercellular junctions was monitored by measuring protein flux across Transwell filters. Time course experiments demonstrated that matrix assembly site expression on the basolateral cell surface preceded intercellular junction formation. Transfer of confluent monolayers to calcium‐free medium resulted in the loss of junctions and in an increase in 125I‐70 kD binding from the apical medium. The increased 125I‐70 kD binding resulted from increased access of 125I‐70 kD to basolateral matrix assembly sites and not from the relocation of binding sites to the apical membrane. To determine the effect of matrix composition on matrix assembly site expression and localization, cells were seeded onto vitronectin‐ or fibronectin‐coated substrates. Fibronectin increased the expression of matrix assembly sites on the apical surface within 24 hours. By 48 hours, matrix assembly sites were located only on the basolateral surface. Vitronectin had no effect on the expression or localization of matrix assembly sites. These results indicate that the expression and localization of matrix assembly sites on the surface of vascular endothelial cells can be regulated by substratum fibronectin. © 1992 Wiley‐Liss, Inc.
A cryptopromoter is activated in the proliferating cell nuclear antigen gene of growth arrested cells
Authors: Sell Christian , Chang Chung‐Der , Koniecki Janice , Chen Hui‐Min , Baserga Renato
Published: February 2005

We have examined the regulation of the proliferating cell nuclear antigen gene (PCNA) in a hamster fibroblast cell line (tk‐ts 13) which is temperature sensitive for growth. These tk‐ts 13 cells, at the restrictive temperature, are growth arrested in the G1 phase of the cell cycle. The cells were stably transfected with a full length human PCNA gene, and the resulting cell lines (K525 cells) were analyzed. We find that, in growth arrested K525 cells, a cryptopromoter is activated in the transfected human PCNA gene. The cryptopromoter resides in intron 4 which is necessary for proper regulation of the PCNA gene. Removal of this intron leads to increased expression of PCNA in cells which have entered the Go state. An Alu sequence residing in intron 4 is implicated as the promoter element which is active during growth arrest. © 1992 Wiley‐Liss, Inc.
Platelet‐activating factor stimulates phospholipase C activity in human endometrium
Authors: Ahmed A. , Smith S. K.
Published: February 2005

Human preimplantation embryos secrete platelet‐activating factor (PAF), which stimulates prostaglandin E2 synthesis from secretory endometrium. This study investigated the action of PAF on phosphatidylinositol 4,5‐bisphosphate (Ptdlns(4,5)P2)‐specific phospholipase C activity in human endometrium. Slices of normal endometrium were incubated with 5 μCi/ml myo‐[2‐3H] inositol for 3 h at 37°C in 95% O2 and 5% CO2 to label tissue phosphoinositides. Inositol phosphates were extracted using trichloroacetic acid precipitation and diethylether neutralization and production was measured using Dowex 1‐X8 anion‐exchange column chromatography. PAF induced rapid and concentration‐dependent accumulation of inositol phosphates (IP) from secretory endometrium, but had no effect on endometrium removed in the proliferative phase of the menstrual cycle. The IP3 fraction was significantly elevated from a median value of 14.0 c.p.m. mg−1 dry wt [range: 8–41 c.p.m. mg−1 dry wt] to 28.0 c.p.m. mg−1 dry wt [range: 11–87 c.p.m. mg−1 dry wt, P < 0.002] following 1 min exposure of secretory endometrium to PAF‐acether, in the presence of 10 mM LiCl. PAF‐induced hydrolysis of Ptdlns(4,5)P2 was inhibited by the specific PAF receptor antagonist WEB 2086, in a dose‐dependent manner (P < 0.02), indicating that in human endometrium Ptdlns(4,5)P2 hydrolysis is mediated via a PAF receptor. These results indicate that PAF receptor coupling activates endometrial Ptdlns(4,5)P2‐specific phospholipase C only in the secretory phase of the menstrual cycle, suggesting that the PAF response may be under ovarian steroid regulation. It is proposed that the ability of the endometrium to respond to PAF appears to be a feature of the preparation of this tissue for implantation and that the second messengers generated may play a role in cellular processes involved in the maternal recognition of very early human pregnancy. © 1992 Wiley‐Liss, Inc.
IL‐3 stimulated haemopoietic stem cell proliferation: Evidence for G protein independent mitogenic signalling events
Authors: Akinsanya Ayodele A. , Whetton Anthony D.
Published: February 2005

Interleukin‐3 stimulates the survival and proliferation of the FDCP‐Mix 1 multipotent stem cell line. We have investigated the possible involvement of a guanyl nucleotide regulatory (G) protein(s) in the IL‐3 stimulated proliferative response. We report here that pertussis toxin (PT) can partially inhibit IL‐3 stimulated DNA synthesis and that this inhibition is bypassed by TPA. The ADP‐ribosylation of the PT substrate G protein in vivo is complete in 2 hours without concomitant inhibition of IL‐3 stimulated hexose transport or Na+/H+ exchange. When loaded into FDCP‐Mix 1 cells fluoroaluminate and GTP‐γ‐S, which can directly activate G proteins, are not capable of mimicking the effects of IL‐3. Evidence is also presented that IL‐3 does not stimulate a membrane‐bound high affinity GTPase activity in the FDCP‐Mix 1 cell line. These data suggest that a PT substrate G protein(s) can influence the IL‐3 signalling cascade in an indirect or permissive manner, but that the IL‐3 receptor does not directly couple to a PT substrate G protein. © 1992 Wiley‐Liss, Inc.
Characterization of metformin transport system in NIH 3T3 cells
Authors: Khan Naim A. , Wiernsperger Nicolas , Quemener Veronique , Havouis René , Moulinoux Jacques P.
Published: February 2005

The biochemical properties of the metformin transport system were studied in NIH 3T3 cells. 14C‐metformin uptake appeared to be a sodium dependent process. Iso‐osmotical replacement of Na+ by choline chloride in the assay medium resulted in a decrease of metformin uptake. Amiloride (200 μM) inhibited the metformin transport by 35% in these cells. Gramicidin, a channel ionophore, was the most effective in inhibiting the metformin transport as compared to valinomycin, a mobile ion carrier, and Ca2+ ionophore (A 23187). Loading of cells with asparagine, ornithine, or polylysine did not influence the uptake process. However, the addition of lysine or arginine significantly stimulated the metformin uptake by NIH 3T3 cells. Similarly, the addition of metformin stimulated the arginine uptake by these cells, suggesting that metformin shares the y+ transport system. Metformin inhibited competitively the uptake of 14C‐spermidine, a molecule of the polyamine family, by NIH 3T3 cells, whereas the latter failed to influence the uptake of the former significantly by these cells. Incubation of NIH 3T3 cells in the presence of difluoromethyl‐ornithine (a suicidal inhibitor of polyamine biosynthesis) stimulated the spermidine, but not the metformin, uptake by these cells. Interestingly, a prolonged incubation of these cells in the presence of metformin failed to down‐regulate the spermidine transport process. The spermidine‐ and methylglyoxal‐bis(guanylhydrazone), MGBG‐transport deficient (3T3MG) cells which do not accumulate exogeneous spermidine or MGBG, took up 14C‐metformin. However, 14C‐metformin uptake by 3T3MG cells was lower than that by normal NIH 3T3 cells. © 1992 Wiley‐Liss, Inc.
Distinct mechanisms of zinc uptake at the apical and basolateral membranes of Caco‐2 cells
Authors: Raffaniello Robert D. , Lee Shih‐Yu , Teichberg Saul , Wapnir Raul A.
Published: February 2005

Zinc uptake mechanisms at the apical and basolateral membrane borders of caco‐2 cells were examined. This human‐derived cell line possesses many morphological and functional characteritics of absorptive small intestinal cells. By day 14, coniluent and well‐differentiated monolayers were formed when the cells were grown on porous polycarbonate filters. Labelled zinc was placed on the apical or basal side of the monolayer and its uptake by the cells, as well as its transport across the monolayer, were measured. Zinc uptake by the cells from the apical side was found to be a saturable process (Kt = 41 μM; Vmax = 0.3 nmols/cm2/10 min) with a diffusional term at higher concetrations (1.0 sec/cm). Apical uptake was not affected by metabolic inhibitors or potential zinc ligands. Zinc uptake from the basolateral side was concentration dependent (Kd = 1.3 sec/cm) and was partially inhibited (30%) by ouabain and vanadate, suggesting that the (Na‐K)‐ATPase on the basolateral membrane is involved in the serosal uptake of zinc by the cell. Transport of zinc across the monolayers from the apical or basolateral compartment was concentration dependent and was not affected by metabolic inhibitors. Zinc transport from the basolateral side was >2‐fold greater than apical transport. Hence, separate mechanisms can be distinguished with respect to zinc uptake at the apical and basolateral membranes of caco‐2 cells. © 1992 Wiley‐Liss, Inc.
Insulin‐like growth factor‐I (IGF‐i) stimulates protein synthesis and collagen gene expression in monolayer and lattice cultures of fibroblasts
Authors: Gillery Philippe , Leperre Armelle , Maquart François‐Xavier , Borel Jacques‐Paul
Published: February 2005

Fibroblasts cultivated in three‐dimensional tissue‐like matrices are characterized by a slowed metabolism and a decrease of protein synthesis, unless they are submitted to physical tensions. We checked the effects of insulin like growth factor‐I (IGF‐I), known as a potent stimulator of mitogenesis and protein synthesis for many cell types, in various models of cultures: confluent monolayers, collagen lattices, non‐retracting or retracting fibrin lattices. IGF‐I (1–100 ng · ml−1) had no effect on cell divisions in lattice cultures. It was able to stimulate collagen lattice retraction when the medium was supplemented with low concentrations of serum. IGF‐I at 10 or 100 ng · ml−1 stimulated collagen and non‐collagen syntheses in all culture systems, but stimulation of collagen synthesis only began at the highest concentration (100 ng · ml−1) in retracted lattices. Northern blot and dot‐olot analyses of mRNAs extracted from monolayer cultures of fibroblasts showed that IGF‐I stimulated pro α1(I) collagen synthesis at the pretranslational level. Cycloheximide (7.5 μg · ml−1) completely inhibited pro α1(I) collagen gene expression induced by IGF‐I. These results show that IGF‐I is a potent stimulus for protein synthesis and collagen gene expression in monolayers and tridimensional cultures of fibroblasts, but that it exerts no mitogenic activity in tridimensional lattices. Synergistic associations of IGF‐I with other growth factors will have to be found in order to reverse the quiescent status of fibroblasts in lattices. © 1992 Wiley‐Liss, Inc.
Transforming growth factor‐β1 inhibitory effect of platelet‐derived growth factor‐‐induced signal transduction on human bone marrow fibroblasts: Possible involvement of protein phosphatases
Authors: Fontenay Michaëla , Bryckaert Marijke , Tobelem Gérard
Published: February 2005

Transforming growth factor‐β1 (TGF‐β1) is a potent growth inhibitor for many cell types. On fibroblasts, TGF‐β1 has been shown to inhibit human platelet‐derived growth factor (PDGF)‐induced mitogenicity. The mechanism implicated in this growth inhibition is unknown. In this work, we show on human bone marrow fibroblasts that TGF‐β1, which inhibited PDGF‐BB mitogenicity, was able to block PDGF‐BB‐induced early events such as polyphosphoinositide (Ptdlns 4,5‐P2, Ptdlns 4‐P, and Ptdlns) breakdown and Ins 1,4,5‐P3 formation. No significant modification by TGF‐β1 of PDGF‐BB binding (n1 = 200,000 vs. n2 = 195,000 sites per cell with TGF‐β1; Kd1 = Kd2 = 0.5 × 10−9M) and of internalization kinetics was observed. In addition, TGF‐β1 was shown to inhibit PDGF‐BB receptor autophosphorylation either in intact cells or in partially isolated membranes and to partially inhibit PDGF‐R tyrosine kinase activity. Since a dephosphorylation mechanism through protein phosphatases could be implicated, we used okadaic acid, a potent inhibitor of type 1 and 2A serine/threonine phosphatases and showed that okadaic acid restored PDGF‐receptor autophosphorylation on tyrosine residues. Based on these data, we suggest that an alternative regulatory mechanism of PDGF tyrosine phosphorylation seems to involve serine/threonine phosphatase activation. © 1992 Wiley‐Liss, Inc.
Brefeldin A, thapsigargin, and AlF stimulate the accumulation of GRP78 mRNA in a cycloheximide dependent manner, whilst induction by hypoxia is independent of protein synthesis
Authors: Price Brendan D. , Mannheim‐Rodman Laura A. , Calderwood Stuart K.
Published: February 2005

The glucose regulated proteins (GRPs) are major structural components of the endoplasmic reticulum (ER) and are involved in the import, folding, and processing of ER proteins. Expression of the glucose regulated proteins (GRP78 and GRP94) is greatly increased after cells are exposed to stress agents (including A23187 and tunicamycin) which inhibit ER function. Here, we demonstrate that three novel inhibitors of ER function, thapsigargin (which inhibits the ER Ca2+‐ATPase), brefeldin A (an inhibitor of vesicle transport between the ER and Golgi) and AlF−4, (which inhibits trimeric G‐proteins), can increase the expression of both GRP78 and 94. The common characteristic shared by activators of GRP expression is that they disrupt some function of the ER. The increased levels of GRPs may be a response to the accumulation of aberrant proteins in the ER or they may be increased in response to structural/functional damage to the ER. The increased accumulation of GRP78 mRNA after exposure of cells to either thapsigargin, brefeldin A, AlF−4, A23187, or tunicamycin can be blocked by pre‐incubation in cycloheximide. In contrast, accumulation of GRPs after exposure to hypoxia was independent of cycloheximide. In addition, the protein kinase inhibitor genistein blocked the thapsigargin induced accumulation of GRP78 mRNA, whereas the protein phosphatase inhibitor okadaic acid caused increased accumulation of GRP78 mRNA. The data indicates that there are at least 2 mechanisms for induced expression of GRPs, one of which involves a phosphorylation step and requires new protein synthesis (e.g., thapsigargin, A23187) and one which is independent of both these steps (hypoxia). © 1992 Wiley‐Liss, Inc.
β1 integrins isolated from embryonic chicken fibroblasts bind to monomers and polymers of type I collagen
Authors: Potts A. Jeannette , Little Charles D.
Published: February 2005

The avian integrin β1 subfamily consists of multiple α‐β subunit heterodimers. We employed two different physical states of type I collagen, monomers and fibrils, in the isolation and characterization of avian collagen integrins. Affinity chromatography showed that three integrins, tentatively designated α155β1 (band 1), α5aβ1, and α3β1 (band 2), bind fibrillar and monomeric collagen under physiological ionic conditions and require divalent cations for binding activity. Sodium chloride gradients (0–0.5 M) were used to assess the functional ability of the integrins to remain bound to the two forms of type I collagen. The results show that integrins elute from the two forms of collagen with distinct fractionation profiles. One integrin, α155β1, binds fibrillar collagen with relatively higher affinity than the other β1 receptors. This same avian integrin, α155β1, is immunoreactive with an antiserum (Hynes et al., 1989) raised against a peptide that corresponds to the entire α5 cytoplasmic domain, and coincidently, part of the α6 cytoplasmic domain (de Curtis et al., 1991). Cell biological studies employing double immunofluorescence show that integrins recognized by this antiserum co‐localize with extracellular deposits of type I collagen. © 1992 Wiley‐Liss, Inc.
Characterization of a simian virus 40‐transformed human podocyte cell line producing type IV collagen and exhibiting polarized response to atrial natriuretic peptide
Authors: Ardaillou Nicole , Lelongt Brigitte , Turner Neil , Piedagnel Rémi , Baudouin Béatrice , Estrade Simone , Cassingena Roland , Ronco Pierre M.
Published: February 2005

Biology of glomerular visceral epithelial cells (“podocytes”) and their role in inflammatory process remain obscure, partly because of the lack of well‐differentiated podocyte cultures. We have established a human cell line by transfecting with a replication‐defective SV40 plasmid (pSVHB1), a primary culture of podocytes derived from an enriched preparation of unencapsulated glomeruli free of tubule and Bowman's capsule contaminants. Podocyte specificity of the primary culture was assessed by a dual immunomorphological and functional approach. The resulting cell line (HGVEC. SV1) was cloned and the clonal cells were adapted to hormonally defined medium supplemented with only 2% newborn bovine serum. Clone A4 has been exhibiting over 35 passages, a combination of markers unique to podocytes, including expression of vimentin, podocalyxin, ectoenzymes (CALLA antigen and mRNA), heparan‐sulfate proteoglycans (molecular mass of core protein = 75 kDa), and production of type IV collagen (α1 and α5 chains) established by immunoprecipitation and Northern blot analysis. Cytokeratin was detected in rare cellular foci and the search of Von Willebrand factor was negative. This clonal cell line has been used to demonstrate: (1) that human podocytes are highly sensitive to atrial natriuretic peptide (ANP) which induced a dose‐dependent increase in cGMP production (x20 at 0.5 μM ANP), and (2) that secretion of ANP‐stimulated cGMP is dramatically polarized as 93% of extracellular cGMP were released in the apical medium when filter‐grown HGVEC. SV1A4 cells were stimulated at their basal pole. © 1992 Wiley‐Liss, Inc.
Interleukin 6 promotes epithelial migration by a fibronectin‐dependent mechanism
Authors: Nishida Teruo , Nakamura Masatsugu , Mishima Hiroshi , Otori Toshifumi
Published: February 2005

We investigated the effect of interleukin 6 (IL‐6) on the migration of rabbit corneal epithelial in vitro and on the attachment of dissociated corneal epithelial cells to a fibronectin matrix. When corneal blocks were cultured with IL‐6 for 24 hours, the length of the path of epithelial migration over exposed corneal stroma increased significantly (p < 0.005 at the concentration of 10 ng/ml) in proportion to the concentrations of IL‐6 (0.1–10.0 ng/ml). The addition of antiserum against fibronectin or of GRGDSP abolished the stimulatory effect of IL‐6 on epithelial migration. When corneal epithelial cells were cultured with various concentrations of IL‐6, suspended, and plated on wells coated with fibronectin (10 μg/ml), the number of cells attached to the wells increased in a dose‐dependent manner. The presence of antibody against fibronectin or of GRGDSP during the attachment assay decreased the number of cells attached to the fibronectin matrix, regardless of the fact that the cells had been cultured with IL‐6 or not. IL‐6 stimulated the attachment of corneal epithelial cells to collagen type IV and to laminin matrices. However, the presence of GRGDSP did not affect the cell attachment to collagen type IV and to laminin. These findings strongly indicate that IL‐6 stimulates epithelial migration in the cornea by a fibronectin‐dependent mechanism, presumably the increased expression of fibronectin receptors. © 1992 Wiley‐Liss, Inc.
Electrogenic Na‐independent HCO3 transport in OK cells
Authors: Pastoriza‐Munoz Enrique , Hsiang Fuling , Graber Mark
Published: February 2005

We have shown previously that OK cells recover from an acid load in a medium nominally CO2‐free by extruding H via a Na/H exchanger and a passive H‐conductive pathway. In this work, the regulation of cell pH (pHi) was studied after additon or withdrawal of CO2/HCO3 (5%CO2, 95 mM HCO3, pH = 8) using the fluoroprobe BCECF. In the presence of Na and amiloride to inhibit Na/H exchange, the recovery of pHi after CO2 entry and CO2 exit were found to depend in part on HCO3 entry and exit, respectively. Efflux of H per se also contributed to restoring pHi after CO2 addition, whereas H influx may have played a smaller role to normalize pHi after CO2 removal. DIDS, 0.5mM, significantly inhibited both recovery phases of pHi. Removal of Na failed to inhibit the recovery of pHi after CO2 addition and removal. Cl removal also failed to inhibit pHi recovery after CO2 removal. Cell depolarization in the presence of Na moderately stimulated the pHi recovery rate after CO2 addition whereas it markedly inhibited the normalization of pHi after CO2 removal. Cell depolarization in the absence of sodium had only a slight effect to increase pHi recovery after CO2 addition but markedly prevented the pHi recovery after CO2 removal. These results indicate that OK cells lack Na or Cl‐dependent HCO3 transport systems. The OK cell possesses a novel stilbene‐sensitive electrogenic HCO3 transport system that is involved in the regulation of cell pH. © 1992 Wiley‐Liss, Inc.
Granulocyte‐Macrophage colony‐stimulating factor and steel factor induce phosphorylation of both unique and overlapping signal transduction intermediates in a human factor‐dependent hematopoietic cell line
Authors: Hallek Michael , Druker Brian , Lepisto Eva M. , Wood Kenneth W. , Ernst Timothy J. , Griffin James D.
Published: February 2005

Steel factor (SF), the ligand for the proto‐oncogene c‐kit, acts synergistically with GM‐CSF or IL‐3 to support the growth of normal human hematopoietic progenitor cell. We examined the effect of SF on GM‐CSF or IL‐3 induced proliferation of a human factor‐dependent cell line, MO7. SF supported MO7 cell proliferation as well as IL‐3 or GM‐CSF alone, and its addition dramatically enhanced (three‐to sixfold) maximal GM‐CSF or IL‐3 stimulated proliferation. SF did not increase the number of affinity of cell surface GM‐CSF receptors. We examined several early events of signal tranduction in an effort to elucidate the biochemical mechanisms of synergy of these factors. Since each of these three cytokines is believed to function in part through activation of a tyrosine kinse, we examined their effects on cellular phosphotyrosine containing proteins. Each cytokine induced rapid, transient, and concentration dependent tyrosine phosphorylation of a number of substrates. For GM‐CSF and IL‐3, these phosphoproteins were indistinguishable (150, 125, 106, 93, 80, 79, 73, 44, 42, and 36 kDa), while SF induced major or minor tyrosine phosphorylation of 205, 140–150, 116, 106, 90, 90, 80, 79, 73, 44, 42, 39, 36, 32 kDa phosphoproteins. Two other signal tranduction intermediates known to be phosphorylated and activated by GM‐CSF and IL‐3, the 70–75 kDa Raf‐1 kinase, and p42 mitogen‐activated protein kinase‐2 (MAPK) were also phosphorylated by SF. Combinations of GM‐CSF or IL‐3 with SF did not further increase the phosphorylation of Raf‐1 or p42 MAPK when compared to any of the factors alone. In contrast SF, but not GM‐CSF or IL‐3, induced tyrosine phosphorylation of phospholipase C‐γ (PLC‐γ). These results indicate that SF and GM‐CSF/IL‐3 have partially overlapping effects on early signal tranducing events, as well as striking differences, such as tyrosine phophorylation of PLC‐γ. This cell line should provide a useful model system to investigate the complicated process of hematopoietic growth factor synergy. © 1992 Wiley‐Liss, Inc.
Leukemia inhibitory factor and interleukin‐11 promote maturation of murine and human megakaryocytes in vitro
Authors: Burstein Samuel A. , Mei Rui‐Lian , Henthorn James , Friese Paul , Turner Katherine
Published: February 2005

The growth‐promoting activities of optimally stimulating concentrations of leukemia inhibitory factor (LIF) and interleukin‐11 (IL‐11), a stromal cell‐derived cytokine, on megakaryocytes in liquid marrow cultures were compared to interleukin‐6 (IL‐6), a known megakaryocytic maturation factor. Maximally stimulating concentrations of LIF (25 ng/ml), IL‐11 (10 ng/ml), or IL‐6 alone (10 ng/ml) promoted an 81, 157, and 153% increase, respectively, in acetylcholinesterase (AchE) activity in murine serum‐free cultures compared with controls (n = 5). In combination with 25 U/ml murine interleukin‐3 (IL‐3), LIF, IL‐6, and IL‐11 showed increases, respectively, of 35%, 49%, and 174% in AchE activity compared with IL‐3 alone (n = 4). Flow cytometric analysis of 4‐day‐old cultures showed that LIF alone had minimal effect on megakaryocytic ploidy, whereas IL‐11 and IL‐6 alone markedly augmented high ploidy cells. Enumeration of cells stained for AchE showed that IL‐11 increased the numbers of Mks in comparison to LIF, IL‐6 or controls by up to 59%. Moreover, a twofold increment in Mk number was noted when IL‐11 was used in combination with IL‐3 (compared with either IL‐3 alone of IL‐3 + IL‐6). Flow cytometric ploidy analysis of 8‐day‐old human liquid marrow cultures showed that either LIF, IL‐11, or IL‐6 alone markedly augmented the percentage of 32N cells compared with cultures containing only human IL‐3. The data suggest that LIF and IL‐11 promote murine and human Mk maturation in vitro, although the effect of IL‐11 exceeds that of LIF in mice. Despite the comparable influence of IL‐11 and IL‐6 on Mk ploidy, IL‐11 has the additional characteristic of enhancing the number of Mks, particularly in combination with IL‐3. © 1992 Wiley‐Liss, Inc.
Atypical receptor‐mediated signal transduction events in the EGF‐dependent growth‐inhibited cell line, MDA‐468
Authors: Church Jon G. , Richardson Vernon J. , Lockwood Ann G.
Published: February 2005

It is now generally considered that early signalling from tyrosine kinases that induce mitogenesis is initiated through the formation of heteromeric complexes consisting of the autophosphorylated tyrosine kinase and a number of tyrosylphosphorylated proteins, including phospholipase C‐γ (PLC‐γ) and GTPase activating protein (GAP). However, since much of this work has been performed on proliferative, chimeric cell lines expressing heterologous receptor molecules, we examined the nature of the epidermal growth factor receptor (EGFR) signalling complex formation in the human breast cancer cell line, MDA‐468. This cell line has an amplified, native EGFR gene, correspondingly overexpresses the EGFR, and its growth in culture is inversely related to the EGF concentration. Our results indicate that in MDA‐468 cells, both the EGFR and PLC‐γ are phosphorylated on tyrosine residues and can be co‐immunoprecipitated. This occurs at both high and low EGF concentrations regardless of the proliferative endpoint. The molecular association is correlated with a significant increase in total inositol phosphates formed in response to the growth factor treatment. In contrast, however, there is no evidence that GAP is either phosphorylated on tyrosine residues or forms a complex with the activated EGFR in EGF‐treated MDA‐468 cells. These observations suggest that as a model for growth factor action, the formation of heteromeric protein signalling complexes may demonstrate considerable diversity depending upon both cell type and physiology. © 1992 Wiley‐Liss, Inc.