bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of murine megakaryocyte colony size and ploidy: Effects of interleukin‐3
Authors: Segal Gerald M. , Stueve Terri , Adamson John W.
Published: February 2005

Megakaryocytes develop from diploid precursor cells that, after variable numbers of mitoses, cease cell division and then undergo synchronous nuclear endoreduplication (endomitosis). Megakaryocyte colony formation represents an in‐vitro model of these processes in which the number and ploidy of colony cells reflect the activity of the mitotic and endomitotic pathways, respectively. We have analyzed the size and ploidization of murine megakaryocyte colonies grown in agar and examined the influence of interleukin‐3 (IL‐3) on these parameters. Colonies were identified in situ by staining for acetylcholinesterase and the ploidy of colony cells was determined by fluorescence cytophotometry. More than 98% of the megakaryocytes that developed in culture could be analyzed. In cultures of unfractionated marrow cells stimulated by either pokeweed mitogen‐stimulated spleen cell‐conditioned medium (PWM‐SCM, a crude source of megakaryocyte colony‐stimulating activity) or IL‐3, the modal ploidy of day‐5 colony megakaryocytes was 16N (range 2N‐128N). The distribution of colony size was described by an inverse exponential relationship. Colony size and geometric mean ploidy were inversely correlated under conditions of maximal stimulation with PWM‐SCM and at all concentrations of IL‐3 tested. Increasing concentrations of IL‐3 in cultures of either unfractionated marrow cells or nonadherent T‐lymphocyte‐depleted (NATD) marrow cells stimulated similar dose‐dependent increases in the mean size and numbers of megakaryocyte colonies but did not significantly alter their ploidy distribution. However, the mean ploidy of colony megakaryocytes in IL‐3‐stimulated cultures of NATD marrow cells was significantly less (P < 0.001) than the mean ploidy of megakaryocytes in IL‐3‐stimulated cultures of unfractionated marrow cells. The mean ploidy of megakaryocytes, which developed in PWM‐SCM‐stimulated cultures, was not affected by initial accessory cell depletion. We conclude that the size and ploidy characteristics of day‐5 murine megakaryocyte colonies are structured as continua and that IL‐3 stimulates an increase in mean colony size and numbers without affecting ploidization. T‐lymphocytes and adherent cells elaborate an activity which promotes endomitosis in vitro; factors in PWM‐SCM can substitute for this activity.
Platelet‐derived growth factor or basic fibroblast growth factor induce anchorage‐independent growth of human fibroblasts
Authors: Palmer Helen , Maher Veronica M. , McCormick J. Justin
Published: February 2005

Anchorage‐independent growth, i.e., growth in semi‐solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage‐independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage‐independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage‐independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet‐derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage‐independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF‐β) decreased the growth induced by PDGF. EGF combined with TGF‐β induced colony formation in semi‐solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage‐independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF‐β can stimulate anchorage‐independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.
Superoxide dismutase activities of differentiating clones from an immortal cell line
Authors: Oberley Larry W. , Ridnour Lisa A. , Sierra‐Rivera Elaine , Oberley Terry D. , Guernsey Duane L.
Published: February 2005

We have studied superoxide dismutase (SOD) levels in the X‐REF‐23 rat embryo fibroblast cell line. X‐REF‐23 is an immortal cell line that maintains a nontransformed phenotype throughout its known lifespan. Low‐passage X‐REF‐23 cells undergo spontaneous differentiation into muscle and adipose cells, while high‐passage X‐REF‐23 cells undergo little or no differentiation. SOD activities were measured in subclones of X‐REF‐23, which differentiate into muscle (AMC subclone) or adipose (AMB‐J) cells, as well as the parental nondifferentiating X‐REF‐23 cells. Total SOD activity increased in all three cell lines with time in culture. Cu‐ZnSOD was induced in the AMB‐J and the X‐REF‐23 cells with time in culture, whereas the AMC cells showed no induction. MnSOD activity was induced during the time period in which differentiation occurred in the two differentiating clones. In contrast, MnSOD was not induced in this time period in the nondifferentiating X‐REF‐23 cell line. However, MnSOD activity was induced in the latter cell line at a much later time. Levels of immunoreactive MnSOD correlated quite well with MnSOD activity in all three cell lines. The nondifferentiating X‐REF‐23 cells, but not the two differentiating cells lines, showed a large increase in cell organelles with time in culture. In particular, an increase in very small mitochondria was observed; these mitochondria often showed evidence of disorganization.
Simultaneous stimulation of urokinase and tissue‐type plasminogen activators by phorbol esters in human ovarian carcinoma cells
Authors: Band Vimla , Karlan Beth Y. , Zurawski Vincent R. , Littlefield Bruce A.
Published: February 2005

OVCA 433 human ovarian carcinoma cells secrete both mammalian plasminogen activators (PAs) urokinase (UK) and tissue‐type PA (tPA). Treatment of cells with 4β‐phorbol‐12‐myristate‐13‐acetate (PMA), a stimulator of protein kinase C (PKC), leads to large increases in the secretion rates of both PA types. PA stimulation by PMA is time‐ and concentration‐dependent, with maximal effects occurring between 12 and 24 h at PMA concentrations of 1–10 ng/ml. The PMA effect is mimicked by mezerein, another known PKC stimulator, but not by 4α‐phorbol or 4α‐phorbol‐12,13‐didecanoate, two phorbol compounds that do not stimulate PKC. PA activity is virtually unaffected by 1‐oleoyl‐2‐acetylglycerol (OAG), a synthetic diacylglycerol that stimulates PKC in vitro but has variable effects on whole cells. PMA stimulation of PA activity is blocked by both actinomycin D and cycloheximide, indicating requirements for new RNA and protein synthesis. When analyzed individually, the relative PMA‐induced increases in UK and tPA activities are identical. Increased UK activity is fully accounted for by increased UK antigen secretion, whereas increased tPA secretion accounts for only about one‐half of the increased tPA activity. Similarly, PMA induces large increases in steady‐state UK mRNA levels, while its effects on tPA mRNA levels are only modest. Thus, while increases in secretion rates and mRNA levels can completely account for UK stimulation, other mechanisms augmenting these processes must exist specifically for tPA. Since the relative increases in UK and tPA activities are identical despite the probable existence of multiple mechanisms contributing to tPA regulation, our data suggest the possibility of interrelationships between the two pathways such that equivalent degrees of UK and tPA activity stimulation are ultimately achieved.
Adenovirus infection retards ribosomal RNA processing
Authors: Lawler Susan H. , Jones Robert W. , Eliceiri Brian P. , Eliceiri George L.
Published: February 2005

Eight hours after infection of KB cells with adenovirus type 12, the rate of conversion from the 32S ribosomal RNA (rRNA) precursor to mature 28S and 5.8S rRNA decreased. An additional RNA species was detected, which appears to be novel, on the basis of its estimated size (about 6.5 kilobases) and its high level of radiolabeling early after infection at low multiplicity.
Selective release from cultured mammalian cells of heat‐shock (stress) proteins that resemble glia‐axon transfer proteins
Authors: Hightower Lawrence E. , Guidon Peter T.
Published: February 2005

Cultured rat embryo cells were stimulated to rapidly release a small group of proteins that included several heat‐shock proteins (hsp110, hsp71, hscp73) and nonmuscle actin. The extracellular proteins were analyzed by two‐dimensional polyacrylamide gel electrophoresis. Heat‐shocked cells released the same set of proteins as control cells with the addition of the stress‐inducible hspllO and hsp71. Release of these proteins was not blocked by either monensin or colchicine, inhibitors ofthe common secretory pathway. A small amount of the glucose‐regulated protein grp78 was externalized by this pathway. The extracel‐lular accumulation of these proteins was inhibited after they were synthesized in the presence of the lysine analogue aminoethyl cysteine. It is likely that the analogue‐substituted proteins were misfolded and could not be released from cells, supporting our conclusion that a selective release mechanism is involved. Remarkably, actin and the squid heat‐shock proteins homologous to rat hsp71 and hsp110 are also among a select group of proteins transferred from glial cells to the squid giant axon, where they have been implicated in neuronal stress responses (Tytell et al.:Brain Res., 363:161‐164, 1986). Based in part on the similarities between these two sets of proteins, we hypothesized that these proteins were released from labile cortical regions of animal cells in response to perturbations of homeostasis in cells as evolutionarily distinct as cultured rat embryo cells and squid glial cells.
Interleukin 3‐stimulated proliferation is sensitive to pertussis toxin: Evidence for a guanyl nucleotide regulatory protein‐mediated signal transduction mechanism
Authors: Kelvin David J. , Shreeve M. , McAuley C. , McLeod D. L. , Simard G. , Connolly J. A.
Published: February 2005

Interleukin 3 (IL‐3) stimulates several biochemical and biological responses in IL‐3‐dependent tissue culture cells. We examined the possibility that guanyl nucleotide regulatory (G) proteins may transduce signals from IL‐3 receptors. We report here that pertussis toxin (PT), which can covalently modify a subclass of G proteins, is capable of inhibiting IL‐3‐stimulated proliferation in a dose‐dependent fashion. PT inhibiton of IL‐3‐stimulated proliferation could be overcome by using the Ca+ + ionophore A23187 in conjunction with TPA. PT could also inhibit IL‐3‐stimulated hexose transport. In the absence of IL‐3, hexose transport could be stimulated by introducing GTPγS into intact cells. From these data we propose that IL‐3 receptors transduce signals via a PT‐sensitive G protein(s).
Regulation of expression of the cell adhesion receptors, integrins, by recombinant human interleukin‐1β in human osteosarcoma cells: Inhibition of cell proliferation and stimulation of alkaline phosphatase activity
Authors: Dedhar Shoukat
Published: February 2005

Recombinant human interleukin‐1β, a mediator of osteoblastic cell function, was found to regulate the expression of the cell adhesion receptors, integrins, on human osteosarcoma cells. Interleukin‐1β (IL‐1β) at picomolar concentrations, specifically elevated approximately six‐ to tenfold the expression of the β1 subunit and its associated α subunits, but not the related vitronectin receptor, within 20 hours. Integrin β1 messenger RNA levels were elevated within 6 hours and peaked to tenfold higher levels after 20 hours exposure to IL‐1β in two human osteosarcoma cell lines. The increase in the cell‐surface β1 integrins resulted in a stronger binding of the IL‐1β‐treated cells to fibronectin. Cell growth was also inhibited by IL‐1β cell morphology was altered, and IL‐1β‐treated cells expressed an approximately two‐ to threefold higher alkaline phosphatase. This increase in alkaline phosphatase activity was found to be independent of the inhibition of cell proliferation. These data indicate that the β1 integrin family of cell surface receptors is a target for regulation by IL‐1β which also regulates cell proliferation and the expression of the osteoblastic phenotype in human osteosarcoma cells.
Analysis of the multiple 5′ and 3′ termini of poly(A) + and poly(A)− deficient thymidylate synthase mRNA in growth‐stimulated mouse fibroblasts
Authors: DeWille James W. , Harendza Christopher J. , Jenh Chung‐Her , Johnson Lee F.
Published: February 2005

Thymidylate synthase (TS) mRNA content increases about 20‐fold when growth‐stimulated mouse cells progress from the GO/G1 phase into the S phase of the cell cycle. Previous studies, using a cell line in which the TS gene is amplified (LU3‐7), indicated that transcriptional initiation as well as polyadenylation of the mRNA occur at several locations in unsynchronized cells. In the present study, we have used S1 nuclease protection assays to analyze the possible significance of the multiple transcriptional initiation and polyadenylation sites. We found that the same pattern of 5′ and 3′ termini were detected with RNA isolated from the overproducing cells as with RNA isolated from the parental mouse 3T6 cell line, demonstrating that the heterogeneous termini are not a consequence of gene amplification. There was no change in the pattern of 5′ or 3′ termini with either cell line during the progression from G1 phase through S phase in serum‐stimulated cells. Therefore, the increase in TS mRNA content is not the result of differential utilization of the various transcriptional initiation or polyadenylation sites. Analyses of poly(A)− deficient cytoplasmic TS RNA showed that the 5′ termini were the same as those found in poly(A)+ mRNA. However, the 3′ termini were extremely heterogeneous in length. Although some of the poly(A)− deficient RNA extended beyond the normal site of polyadenylation, most of it was shorter than full‐length TS mRNA.
Identification of a 15,000‐molecular‐weight form of immunoreactive transforming growth factor α in extracts of porcine pituitary
Authors: Riss Terry L. , Sirbasku David A.
Published: February 2005

Two different mitogenic activities were identified from extracts of porcine pituitary by using COMMA‐D mouse mammary epithelial cells in a serum‐free 3H‐thymidine incorporation assay. Porcine pituitaries were extracted in phosphate‐buffered saline (pH 7.4) and 25–80% (NH4)2SO4 pellets were dialyzed and chromatographed by using DEAE‐Sepharose Chromatography (pH 8.0), resulting in two peaks (I and II) of mitogenic activity. Peak I represented a recovery of 73% of the units of mitogenic activity present in crude extract of pituitary while only 1.25% of the activity was recovered in peak I was further purified by using CM‐Sephadex and heparin‐Sepharose chromatographies and yieled a mitogen that was able to elicit one‐half‐maximal stimulation of 3H‐thymidine incorporation by COMM‐D cells at 48 pg/ml. As expected with pituitary as the tissue source, peak I was confirmed to be basic fibroblast growth factor (bFGF) by using specific antibodies in enzyme‐linked immunosorbent assay and Western immunoblotting pocedures. Peak II was futher purified by using chromatorofocusing (pH 7.3–5.0), reverse‐phase, and action‐exchange HPLCs. The mitogenic activity eluted at pH 6.3 from chromatofocusing, migrated as a 13‐kDa molecule on gel filtration HPLC, and did not bind to heparin‐Sepharose under conditions which bound fibroblast growth factors. The material purified from peak II and rat synthetic transforming growth factor α (TGFα) competed in a parallel fashion with 125I‐epidermal growth factor for receptor binding with A431 human epidermal carcinoma cells. In addition, the mitogen purified from peak II showed a single immunoreactive band migrating at 15 kDa when specific antiserum against TGfα was used in a Western immunoblotting procedure. The data suggest that in addition to the well‐documented presence of bFGF, normal adult porcine pituitaries contain a 15‐kDa form of immunoreactive TGFα that binds to EGF receptors and is mitogenic for mammary epithelial cells.
Developmental regulation of calcium‐binding proteins (calelectrins and calpactin I) in mammary glands
Authors: Lozano Joseph J. , Silberstein Gary B. , Hwang Soo‐In , Haindl Alfred H. , Rocha Victor
Published: February 2005

We recently showed that mammary glands contain a novel class of calcium‐binding proteins (CBPs) that bind to membranes in a calcium‐dependent manner. We have also established that these mammary CBPs are equivalent to the calelectrins and calpactin I/p36. Since it has been suggested that these proteins might be involved in exocytosis, we examined mammary glands for these CBPs during secretory differentiation. Immunohistochemical examination showed, glands from virgin animals to be rich in calelectrins and calpactin I/p36, while glands from lactating animals contained little immunoreactive material. In addition, silver‐staining and immunoblot estimation of the CBPs in lysates from collagenase harvested secretory epithelia showed these proteins to be significantly reduced compared to nonsecretory epithelia. Close examination of the CBP immunoreactive cells of the mammary gland shows that ductal cells are prominent in their staining and that the immunoreactive material is associated with the cell surface. Also, in juvenile glands the.myoepithelial stem cells (cap cells) of the elongating end bud are devoid of the CBPs. In contrast to the in vivo data, epithelia cultivated on collagen gels demonstrate comparable levels of the CBPs in both nonsecretory and secretory monolayers. The in vivo data indicate that the CBPs are developmentally regulated during mammary gland differentiation such that secretory epithelia are essentially devoid of these novel proteins. Furthermore, a role for calelectrin and calpactin I/p36 in exocytotic casein secretion is questioned.
Effect of macrophages on the translocation of protein kinase C in concanavalin a‐stimulated lymphocytes
Authors: Grove Deborah S. , Mastro Andrea M.
Published: February 2005

The concanavalin A (Con A)‐induced proliferation of lymph node lymphocytes is dependent on the presence of macrophages. When lymphocytes are depleted of macrophages, Con A is no longer mitogenic. Either 12‐0‐tetradecanoylphorbol‐13‐acetae (TPA), interleukin 1 (IL1), or macrophages in combination with Con A can restore proliferation. To establish where the proliferation process is blocked in the absence of macrophages, an early step in the signalling pathway, the activation of protein kinase C, was examined. It was found that although Con A caused translocation of protein kinase C from the cytosol to the membrane of lymph node cells, when the lymph node cells were depleted of macrophages and exposed to Con A, this translocation of protein kinase C did not occur. Instead, protein kinase C activity decreased in the membrane fraction and increased in the cytosol. On the other hand, TPA caused translocation of protein kinase C (PKC) from the cytosol to the membrane regardless of the presence of macrophages. However, the macrophage product, IL1, alone or in combination with Con A did not cause translocation of protein kinase C. In a reconstitution experiment, in which lymph node cells were depleted of macrophages and then macrophages were added back, the addition of Con A again lead to translocation of protein kinase C from the cytosol to the membrane. This combination also restored cell proliferation. Therefore, the Con A induced PKC translocation in T lymphocytes is macrophage mediated. TPA overcomes the macrophage requirement by directly activaing PKC, while IL1 appears to act at a different step in proliferation.
Alteration of microtubule physiology in hepatocytes by insulin
Authors: Caron Joan M.
Published: February 2005

Incubation of primary cultures of rat hepatocytes with insulin caused an alteration in several parameters of microtubule physiology. There was a transient decrease in total cellular tubulin levels and, at the same time, an apparent stabilization of microtubule polymers which was also transient. The level of microtubule polymer, however, was not altered, which indicated that an insulin‐inducible decrease in the level of nonpolymerized tubulin was responsible for the decrease in total cellular tubulin. These changes were followed by an increase in tubulin mRNA levels and, shortly thereafter, by an increase in tubulin protein synthesis. This study demonstrates that microtubule physiology in hepatocytes is modulated by insulin and, further, suggests a model by which insulin‐induced changes in microtubule physiology may play a role in regulating other insulin responses.
Mutual contact of murine erythroleukemia cells activates depolarizing cation channels, whereas contact with extracellular substrata activates hyperpolarizing Ca2+‐dependent K+ channels
Authors: Arcangeli Annarosa , Del Bene Maria Riccarda , Poli Riccardo , Ricupero Letizia , Olivotto Massimo
Published: February 2005

This study deals with the modulation of the plasma membrane potential (Δψp) of murine erythroleukemia (MEL) cells by cell‐substratum or cell‐cell contact. Δψp was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane; it appeared strongly, and inversely, influenced by the two types of cell contacts. Contact with the culture surface produced a Δψp hyperpolarization directly proportional to average distance among the ideal centers of the cells on this surface (d) within the range 10–80 μm. A detailed mathematical analysis of the function Δψp = f(d) is presented, as well as experiments involving the use of ionophores (valinomycin and A23187) and the conditioning of the culture surface. We concluded that the d‐dependent hyperpolarization (dDH) was the result of a complex interplay between the activating properties of substratum on Ca2+‐dependent K+ channels (KCa) and some substratum‐adherent factors that are shed by MEL cells and antagonize KCa activation (substratum‐attached cellular factors = SACF), By contrast, contact of the cells with each other, obtained by incubating MEL cells at d smaller than the average cell diameter (Φ = 10 μm), produced a marked Δψp depolarization. This intercellular contact‐dependent depolarization (ICDD) was unaffected by valinomycin; it was abolished by substituting Na+ in the external medium with a nondiffusible cation (choline), which shows that ICDD was sustained by Na+ influxes, probably mediated by stretch‐activated (s.a.) cation channels.
Sphingosine inhibits phorbol 12‐myristate 13‐acetate‐, but not serum‐induced, activation of Na+/H+ exchange in mammalian cells
Authors: Gillies Robert J. , Martinez Raul , Sneider Judith M. , Hoyer Patricia B.
Published: February 2005

Addition of serum to quiescent mammalian cells in culture initiates a series of events which culminates in DNA replication and cell division. One of the earliest events in this sequence of events is activation of Na+/H+ exchange, which can result in an increase in intracellular pH (pHin). The regulation of this change in activity is not known. Since treatment of 3T3 cells with activators of protein kinase C (kinase C) can result in an increased pHin, it has been hypothesized that serum stimulation of kinase C is responsible for activation of Na+/H+ exchange. Recently, sphingolipids have been discovered to inhibit kinase C both in vitro and in vivo. Therefore, we undertook the present study to ask whether or not inhibition of kinase C using sphingolipids prevents mitogen‐induced alkalinization in 3T3 cells. Our results indicate that activators of kinase C stimulate Na+/H+ exchange in normal human fibroblasts (BoGi), but not in mouse embryo (3T3) cells. Addition of serum to BoGi cells, on top of saturating doses of phorbol 12‐myristate 13‐acetate (PMA), results in a further cytoplasmic alkalinization. Furthermore, sphingosine prevents the PMA‐induced increase in pHin in BoGi cells, and phosphorylation of an 80 kDa protein in 3T3 cells, but not the serum‐induced alkalinization in either BoGi or 3T3 cells. These data indicate that activation of kinase C does not participate in the physiological activation of Na+/H+ exchange in human fibroblasts or mouse embryo cells by serum.
Insulin receptor and altered glucose transport in a monensin‐resistant mutant of Chinese hamster ovary cell
Authors: Seguchi Tadashi , Yoshimura Akihiko , Ono Mayumi , Shite Satoshi , Kasahara Michihiro , Ebina Yosuke , Rutter William J. , Kuwano Michihiko
Published: February 2005

A monensin‐resistant mutant Monr‐31, derived from Chinese hamster ovary (CHO) cell line, has been shown to have a reduced number of insulin receptors and a reduction in glucose uptake in response to insulin. We have further investigated the possibility that altered glucose uptake in Monr‐31 cells is related to an alteration in the activity of the insulin receptor. Uptake of glucosamine, 2‐deoxy‐D‐glucose, and 3‐O‐methyl‐D‐glucose in Monr‐31 cells was one‐half to one‐third that of CHO cells. The cellular content of the glucose transporter in Monr‐31 was reduced to about one‐third that of CHO as assayed by use of an antiglucose transporter antibody. After transfection with the human insulin receptor cDNA, we obtained clones CIR‐0 from CHO, and MIR‐2 and MIR‐15 from Monr‐31; CIR‐0 expressed a tenfold higher level of the insulin‐binding activity than did CHO, and MIR‐2 and MIR‐15 expressed a 20‐fold higher level than did Monr‐31. Glucose uptake in both CHO and CIR‐0 was significantly enhanced by exogenous insulin, but not in Monr‐31, MIR‐2, and MIR‐15. The β‐subunits of insulin receptor in CHO, CIR‐0, Monr‐31, and MIR‐2 were similarly phosphorylated. The decreased glucose transport activity in Monr‐31 cells is discussed in relation to the absence or presence of insulin receptor expression.
Interaction of platelet‐activating factor with endothelial and vascular smooth muscle cells in coculture
Authors: Stoll Lynn L. , Spector Arthur A.
Published: February 2005

Platelet‐activating factor (1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine [PAF]) is a vasoactive ether lipid produced by activated blood cells. To examine the molecular traffic and sites of metabolism of PAF released in the vascular wall, we used a coculture system in which endothelial cells are grown on micropore filters suspended over confluent cultures of vascular smooth muscle cells. The endothelial cells took up PAF 5–7 times more readily from the apical than from the basolateral surface, converting it to 1‐O‐alkyl‐2‐acyl‐sn‐glycero‐3‐phosphocholine (2‐acyl‐PAF) and other minor metabolites. Intact endothelial monolayers effectively shielded the underlying smooth muscle cells from PAF present in the apical fluid; after a 30‐min incubation with [3H]‐PAF, only 1% of the radioactivity was transferred to the interstitial fluid. By contrast, PAF readily entered the interstitial fluid when the endothelial monolayers were injured by exposure to xanthine and xanthine oxidase. PAF did not significantly increase the permeability of endothelial monolayers to albumin. Smooth muscle cells took up and metabolized interstitial PAF more quickly and more completely than did endothelial cells; 65% was converted to 2‐acyl‐PAF in 15 min by the smooth muscle cells. PAF enhanced the proliferative effect of PDGF on smooth muscle cells, as assessed by [3H]‐thymidine incorporation. These findings suggest that endothelial cells form a barrier to PAF released at the luminal surface, but PAF released in the vascular intima interacts primarily with smooth muscle cells, possibly stimulating proliferation in these cells.
Differential responses to growth factors by normal human mesothelial cultures from individual donors
Authors: Lechner John F. , LaVeck Moira A. , Gerwin Brenda I. , Matis Elizabeth A.
Published: February 2005

A significant interindividual variation in the growth rates is found in normal cultured human mesothelial (NHM) cells derived from different donors. This variation is observed when the mesothelial cells are incubated in medium containing serum and when the potencies of several separate growth factors are measured by using defined media. Depending on the donor, gamma‐interferon and interleukin‐2 can be toxic, have no effect, or stimulate the growth rate of NHM cells. Cultured NHM cells can be induced to multiply by growth factors that are released by activated macrophages. Thus, interindividual variation in NHM cell growth control could play a role in the pathogenesis of mesothelioma for a person exposed to asbestos.
Cellular interactions between 3T3 cells and interleukin‐3‐dependent multipotent haemopoietic cells: A model system for stromal‐cell‐mediated haemopoiesis
Authors: Yamazaki K. , Roberts R. A. , Spooncer E. , Dexter T. M. , Allen T. D.
Published: February 2005

With the aid of a multipotent stem cell line (FDCP‐mix cells) co‐cultured with either normal or irradiated Swiss 3T3, cellular interactions between stromal cells and haemopoietic stem cells were studied by electron microscopy and time‐lapse video microscopy. When cultured in the presence of interleukin 3 (IL‐3) but in the absence of stromal cells, the FDCP‐mix cells have a characteristic blast morphology. In the absence of IL‐3, the cells die unless they are co‐cultured with marrow stromal cells or 3T3 cells. In the latter case, they attach, proliferate, and differentiate on both normal and irradiated Swiss 3T3 cell layers without the addition of extrinsic growth factor (IL‐3). At the initial attachment sites of these two cell lines, cellular recognition seemed to be mediated by the formation of microvillus cytoplasmic projections and extracellular matrix. These areas may well be the sites of plasma‐membrane‐bound signalling/adhesional molecules between the interacting cells.
Effects of fluorescent derivatives of TPA on HL60 cells: Dissociation between the differentiation‐induced and protein kinase C activity
Authors: Tran Phuong Lan , Le Peuch Christian , Basset Michele
Published: February 2005

The four fluorescent derivatives of TPA—dansylaza‐TPA, NBDaza‐TPA, and (N)‐and (P)‐dansylamino‐TPA—were synthesized and examined for their ability to induce differentiation in human promyelocytic leukemic HL60 cells. At a concentration of 20 nM, all the derivatives inhibited proliferation and induced 60–80% of the cells to differentiate into macrophage‐like cells. Removal of dansylaza‐TPA from the medium after 5 h did not arrest adherence or the expression of nonspecific esterase activity. However, upon removal of any of the other three compounds after 5 h, HL60 cells became nonadherent and expressed low nonspecific esterase activity after additional culture. To investigate the relationship between protein kinase C (PKC) activation and cell maturation, PKC activity and translocation were measured after 0.5, 5, 24, and 48 h of treatment with each compound. Cells induced to differentiate by dansylaza‐TPA or (N)‐ or (P)‐dansylamino‐TPA exhibited enhanced PKC activity, 50–80% of which was located in the particulate fraction. In cells that differentiated with NBDaza‐TPA, 65–70% of PKC activity remained in the cytosol. After removal of the TPA derivatives, all cells exhibited PKC activity in the cytosol. These results indicate that the fluorescent derivatives are as potent as TPA in inducing HL60 cell differentiation. However, in the case of NBDaza‐TPA and (N)‐ or (P)‐dansylamino‐TPA, their continuous presence in the culture medium was required for the recruitment of cells to differentiate. Consequently, it is suggested that activation and translocation of PKC are among the early biochemical events that trigger HL60 cell differentiation. Nevertheless, these two events alone are not sufficient to induce differentiation.
Serum stimulation of quiescent human fibroblasts induces the synthesis of tissue factor mRNA followed by the appearance of tissue factor antigen and procoagulant activity
Authors: Bloem Laura J. , Chen Lisa , Konigsberg William H. , Bach Ronald
Published: February 2005

Expression of the gene for tissue factor, the cell‐surface initiator of blood coagulation, was examined by stimulating growth‐arrested human fibroblasts with serum and measuring changes in the cellular content of tissue factor mRNA, antigen, and activity. Maximum tissue factor mRNA levels were reached within 1 h following serum induction and slowly declined to basal levels from 24 to 48 h after stimulation. The appearance of the tissue factor mRNA was followed by an increase in tissue factor antigen and activity. The parallel rise in antigenically positive protein and procoagulant activity was first observed about 2 h after serum stimulation with a peak at 12 h followed by a slow decline during the next 36 h. The serum‐induced synthesis of the tissue factor mRNA was independent of de novo protein synthesis as demonstrated by the increased tissue factor mRNA levels generated in the presence of cycloheximide. The results of this study suggest that the synthesis of tissue factor in human fibroblasts i s regulated principally at the level of transcription. In one strain of fibroblasts the activity/antigen ratio, during the period of maximum synthesis, was indistinguishable from that of tissue factor which had been immunoaffinity purified from human brain and reconstituted into phospholipid vesicles. However, during serum starvation the activity/antigen ratio in these cells was significantly reduced. Western blot analysis revealed that in serum‐starved cells there was an accumulation of truncated forms of the tissue factor antigen while in the serum‐stimulated cells only the full‐length antigen was observed.
Synchronized human diploid fibroblasts: Progression capabilities of a subpopulation that fails to keep pace with the predominant, rapidly dividing cohort of cells
Authors: Tobey R. A. , Oishi N. , Crissman H. A.
Published: February 2005

Highly synchronized cultures of HSF‐55 human diploid fibroblasts contain subpopulations of cells with intact plasma membranes that do not participate in the parasynchronous division wave. To determine the fate of these laggard cells, cultures were incubated with BrdU for variable periods to label newly replicated DNA in both the readily synchronizable and nonsynchronizable subpopulations. The kinetics of labeling with BrdU were determined with a two‐laser flow cytometric technique that did not employ antibody to BrdU, but instead monitored emission of fluorescence from DNA‐specific stains that differed in the degree of BrdU‐induced quenching of their fluorescence signals. Approximately 90% of the cells rapidly incorporated BrdU and later divided within a 3 hr period. The remaining 10% of the cells, however, were found to reside within a minority subpopulation that maintained the capacity to traverse the cell cycle, but at a greatly reduced rate relative to the progression capacity of the majority of cells. Cells were viably sorted from these cohorts within the synchronized culture, and their kinetic behavior was determined through direct measurement of their growth rates and plating efficiencies. As predicted by the BrdU labeling studies, the sorted cells from the minority, slowly traversing subpopulation divided at a rate that was 30 to 50% lower than that obtained with cells sorted from the readily synchronizable subpopulation. From consideration of the kinetics of entry into S‐phase of the majority and minority subpopulations, protocols are described that should allow preparation of relatively pure populations of both early‐ and late‐replicating species of human DNA.
Effects of biaxial deformation on pulmonary artery endothelial cells
Authors: Gorfien S. F. , Winston F. K. , Thibault L. E. , Macarak E. J.
Published: February 2005

An apparatus has been designed to subject vascular cells grown on a compliant substrate in vitro to uniform, quantifiable levels of biaxial deformation. The system described can be controlled with respect to strain level, rate, and frequency to mimic the pulsatile force to which vascular cells are exposed in vivo under both physiologic and pathologic conditions. In the experiments presented here, bovine pulmonary artery endothelial cells were grown on a substrate of segmented polyurethane urea (MitrathaneTM). Cell growth and morphology on this substrate were compared with those of cells grown on standard tissue culture polystyrene with no difference noted between the two substrates. Primary cultures of pulmonary artery endothelial cells were seeded onto Mitrathane, which was then subjected to cyclic biaxial deformation‐producing strains of 0.78%, 1.76%, 4.9%, or 12.5% at a frequency of 1 sec−1 and a duty cycle of 0.5 sec−1 for 7 h. Cells subjected to deformations generating strains of either 4.9% or 12.5% secreted significantly less fibronectin than nondeformed cells. Similar results were obtained in experiments using cloned pulmonary artery endothelial cells on Mitrathane subjected to the 4.9% strain; however, total protein synthesis was increased. Cell viability and DNA synthesis were not affected by cyclic biaxial deformation in these experiments.
Interaction of serum and colony‐stimulating factor for survival of a factor‐dependent hemopoietic progenitor cell line
Authors: Böhmer Ralph M.
Published: February 2005

The growth, in vitro of the murine myeloid cell line FDC‐P1 depends on the presenc of serum and a murine hemopoietic growth factor (either granulocyte/ macrophage colony‐stimulating factor (GM‐CSF) or multipotential colony‐stimu‐lating factor (multi‐CSF, IL3)). To determine the differential roles of serum and colony‐stimulating factor (CSF) during the growth of FDC‐P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (> 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re‐addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (>> 105/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum‐free conditioned medium from dense cultures made the survival and growth of FDC‐P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.
Growth, enzyme activity, sugar transport, and hormone supplement responses in cells cloned from a pig kidney cell line LLC‐PK1
Authors: Miller John H. , Heath Lisa N.
Published: February 2005

Three clones of the pig kidney cell line LLC‐PK1 were isolated and characterized with regard to morphology, growth, proximal tubule enzyme activity, sugar uptake capacity, and hormone and drug responsiveness in a defined medium. Clone N4 was similar in morphology to the wild type (WT), whereas clone F8 showed loose attachment to the substrate, formed large, sweeping domes, and had an elongated desmosome junction between cells. The third clone, F2, did not form domes and showed a marked reduction in growth rate. Cultures of WT, N4, and F8 had higher specific activities of the enzymes alkaline phosphatase and γ‐glutamyl transpeptidase at confluence relative to growing cells; however, there was no evidence of an increase in activity of either enzyme at confluence in F2. Phlorizin‐sensitive α‐methyl‐D‐glucoside uptake and cytochalasin B‐sensitive 2‐deoxy‐D‐glucose uptake were measured in confluent cultures grown on porous filter supports. None of the clones lacked either of the hexose transport systems, although quantitative differences were evident. N4 cells grown in a defined medium in 96‐well culture plates were tested in situ for their enzyme responses to differentiation inducers, tumor promoters, and hormones. Alkaline phosphatase activity was significantly increased at confluence by serum, parathyroid hormone (PTH), and vasopressin (AVP), and was decreased by tetradecanoylphorbol acetate (TPA) and epinephrine (EPI). Glutamyl transpeptidase activity was decreased at confluence by serum, TPA, and EPI. Similar tests on α‐methyl‐D‐glucoside uptake showed that serum, TPA, PTH, and AVP had no significant effect on phlorizin‐sensitive uptake; however, calcitonin increased uptake by 84% (n = 18). It was concluded that LLC‐PK1 clones maintained in a defined medium are useful models for studying renal cell function.
Regulation of levels of IL‐1 mRNA in human fibroblasts
Authors: Yamato Kenji , El‐Hajjaoui Ziad , Koeffler H. Phillip
Published: February 2005

Interleukin‐1 (IL‐1) has a crucial role in host defenses, inflammatory processes, and tissue homeostasis. A wide variety of cells produce this protein in response to a number of extracellular stimuli including microorganisms, antigenic stimuli, and products from other cells. Regulation of IL‐1 production at the molecular level is poorly understood. We studied expression, intracellular signals, and posttranscriptional regulation of IL‐1 mRNA in human mesenchymal cells by using Northern blot analysis. Tumor necrosis factor alpha (TNFα) and activators of protein kinase C including 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and teleocidin induced the accumulation of IL‐1β mRNA in human fibroblasts (WI‐38). Effect of TNFα was not blocked by inhibitors of either protein synthesis (cycloheximide) or protein kinase C activity. Accumulation of IL‐1β mRNA was also increased by a calcium ionophore (A23187) and an inhibitor of the Na+/K+ pump (ouabain); both compounds are known to increase cytoplasmic levels of Ca++. Stability of IL‐1β mRNA in fibroblasts exposed to TPA was more than fourfold greater than after fibroblasts were exposed to either TNFα or cycloheximide. This suggests that posttranscriptional stabilization of IL‐1β mRNA is a major mechanism leading to accumulation of IL‐1β mRNA after activation of PKC in fibroblasts. Fibroblasts did not express IL‐1α mRNA after exposure to stimuli which induced the accumulation of IL‐1β mRNA. In summary, several different pathways regulate levels of IL‐1β mRNA in human mesenchymal cells.
Role of extracellular matrix in the action of basic fibroblast growth factor: Matrix as a source of growth factor for long‐term stimulation of plasminogen activator production and DNA synthesis
Authors: Flaumenhaft Robert , Moscatelli David , Saksela Olli , Rifkin Daniel B.
Published: February 2005

When bovine capillary endothelial (BCE) cells were treated with 10 ng/ml of basic fibroblast growth factor (bFGF) for 10 or 30 minutes at 37°C, washed extensively with phosphate‐buffered saline (PBS) and incubated in bFGF‐free medium, plasminogen activator (PA) production was stimulated to the same extent as in cells exposed continuously to bFGF. Three methods of removing bFGF from heparin‐like binding sites in the extracellular matrix, but not from bFGF receptors, abolished this long‐term effect of a brief exposure to bFGF. First, BCE cells exposed to bFGF for 30 minutes were washed with 2M NaCI and incubated in bFGF‐free medium. Second, BCE cells were incubated with bFGF for 10 minutes in the presence of heparin, and cells were washed with PBS and incubated in bFGF‐free medium. Third, BCE cell cultures were treated with heparinase and exposed to bFGF. Each of these treatments abolished the long‐term (24‐48 hours) stimulation of PA production normally observed after brief exposure to bFGF. In each of these experiments, incubation of cells in bFGF‐containing medium after the treatments resulted in normal stimulation of PA production, demonstrating that the treatments did not harm the cells. Stimulation of DNA synthesis was observed when cells were exposed to bFGF for 2 hours at 4°C, incubated in bFGF‐free medium for 24 hours at 37°C, and assayed for 3H‐thymidine incorporation. However, no stimulation was observed if the 2 hours incubation at 4°C was carried out in the presence of heparin. Thus, long‐term stimulation of PA activity and DNA synthesis after a brief exposure to bFGF seems to be a consequence of bFGF binding to the extracellular matrix. The extracellular matrixmay act as a physiologic buffer, binding bFGF when concentrations are high and releasing it later for interaction with its receptor. This interaction with matrix may be required for the in vivo action of bFGF.
Maturation‐associated loss and incomplete de novo synthesis of the transferrin receptor in peripheral sheep reticulocytes: Response to heme and iron
Authors: Ahn Jinhi , Johnstone R. M.
Published: February 2005

Hemin, but not iron, in the culture medium stimulates the maturation‐associated loss of the transferrin receptor from sheep reticulocytes (t1/2for loss ∼ 6 hr) and its appearance in a population of externalized vesicles. A similar pattern is seen with nucleoside binding (a measure of the nucleoside transporter), where hemin increases the loss of binding activity from the cells during culture, concomitant with an increase in nucleoside binding in the externalized vesicles. Sheep reticulocytes retain the ability to synthesize the transferrin receptor, but the 35S‐labeled receptors are not detected in released vesicles. Whereas hemin stimulates the loss of 35S‐labeled transferrin receptors from the cell (t1/2 for loss ∼ 20 hr), nonheme iron is more effective than heme. This difference in response of native and 35S‐labeled receptor to hemin and iron supplements appears to be related to the differences in the two classes of receptors. Although the 35S‐labeled receptor binds transferrin and both native and 35S‐labeled peptides comigrate after chemical deglycosylation, the 35S‐receptor is ∼ 2 kD smaller than the native receptor and fails to acquire its complete size even when chased for up to 24 hr. Moreover, the 35S‐labeled receptor is not expressed at the cell surface, but is retained in a nonrecycling compartment, where it is insensitive to digestion by trypsin at both 0°C and 37°C.