bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin‐like growth factor I/somatomedin‐C
Authors: Strain Alastair J. , Hill David J. , Swenne Ingemar , Milner R. D. G.
Published: February 2005

Hepatocytes were isolated by gentle collagenase digestion of liver fragments from human fetuses of 8–16 weeks gestation obtained following prostaglandin‐induced pregnancy terminations. They were maintained on collagencoated tissue culture dishes in selective arginine‐free medium for up to 72 hr, and the action of hormones and growth factors on DNA synthesis was studied by autoradiography following incubation with 3H‐thymidine. The labeling index of hepatocytes was consistently enhanced by 25–250 ng/ml human placental lactogen (HPL), 25–250 ng/ml human growth hormone (HGH), 10–50 ng/ml insulin‐like growth factor l/somatomedin‐C (IGF l/Sm‐C), and 10% dialyzed fetal calf serum, reaching a maximum of three‐ to four‐fold greater than in basal medium alone. Under basal conditions, 30% of hepatocytes stained positively for the presence of IGF peptides using a monoclonal antibody raised against purified human IGF l/Sm‐C. Although this proportion did not change following treatment with HGH and HPL, IGF l/Sm‐C released by cells into culture medium was considerably increased in the presence of both hormones. Incubation with the SmC 1.2 monoclonal antibody abolished the increase in labeling index in response to IGF l/Sm‐C and partially blocked the response to both HPL and HGH. These results indicate that both HPL and HGH stimulate DNA synthesis in human fetal hepatocytes and suggest that this effect is at least partly indirect through the release and paracrine action of IGF l/Sm‐C.
Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: Involvement of heat shock proteins
Authors: Lee Yong J. , Dewey William C.
Published: February 2005

After sodium arsenite (100 μM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one‐dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two‐dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000‐fold increase in survival from 10−6 to 10−1 after 4 hr at 43°C, and as a thermotolerance ratio (TTR) of 2–3 at 10−3 isosurvival for heating at 45.5°C. Cycloheximide (CHM: 10 μg/ml) or puromycin (PUR: 100 μg/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2–6 hr before heating and left on during heating caused a 10,000–100,000‐fold enhancement of survival when cells were heated at 43°C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43°C after CHM treatment were much different than those manifesting resistance to 43°C after arsenite treatment. Arsenite‐treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45°C after 5 hr of heating at 43°C, compared with less thermotolerance (TTR of about 2) for the CHM‐treated cells heated at 45°C after 5 hr of heating at 43°C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43°C or 45.5°C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43°C with little resistance at 45.5°C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45°C caused by heating at 43°C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.
Na+ for H+ exchange in rabbit erythrocytes
Authors: Escobales Nelson , Rivera Alicia
Published: February 2005

The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 μM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pHO = 8.0, in the presence and absence of 60 μM 5,N,N‐dimethyl‐amiloride (DMA). The H+ efflux rate in a K+‐containing medium was 116.38 ± 4.5 mmol/l cell × hr. Substitution of NaO+ for KO+ strongly stimulated H+ efflux to 177.89 ± 7.9 mmol/l cell × hr. The transtimulation of H+ efflux by NaO+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 × 10−7 M. The sequence of substrate selectivities for the external transport site were Na ⋙ Li > choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3−. The DMA sensitive H+ efflux was a saturable function of [Na+]O, with an apparent Km and Vmax of about 14.75 ± 1.99 mM and 85.37 ± 7.68 mmol/l cell × hr, respectively. However, the NaO+‐dependent and DMA‐sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 ± 3.0 mmol/l cell × hr) which was abolished when pHO was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.
Endogenous thiol levels in heterogeneous murine tumor cells as a function of the physiological state and the response to X‐irradiation
Authors: Dethlefsen Lyle A. , Biaglow John E. , Peck Vickie M. , Ridinger David N.
Published: February 2005

The endogenous thiols (PSH, protein sulfhydryls; NPSH, nonprotein sulfhydryls; and GSH, glutathione) were measured in the 66 and 67 murine carcinoma cells growing under different physiological conditions in vitro (e.g., proliferation, P; nutrient‐deprived quiescence Ql; and Ql cells stimulated by refeeding the monolayer in situ and assayed 4 (St4) and 14 (St14) h later). The aerobic radiation response was also studied as a function of the physiological state and thiol concentration. The changes in PSH levels suggest that the proportion of thiol‐containing proteins changed whenever the cells were in transition between different physiological states (e.g., when Ql cells were stimulated by refeeding, the proportion of PSH was elevated dramatically over either Ql or P cells). The NPSH and GSH levels were both down significantly in the Ql vs. P cells as was the total thiol level (PSH plus NPSH). Fourteen h but not 4 h after stimulation, the NPSH and GSH levels had returned to or exceeded the P‐cell levels. Also, the proportion of GSH in the NPSH fraction varied as a function of the physiological state. The 66 and 67 Ql cells were both more radiosensitive than the respective P cells. Also, the 66 cell radiation‐induced cytotoxicity had returned to the P response by about 4 h after refeeding but the stimulatec 67 cells had not. However, no overall correlation was apparent between the various aerobic radiation responses and the pool sizes of either the total thiols or of the various subsets of thiols. The depressed total thiol level and the increased radiosensitivity of the Ql cells could represent a cause‐and‐effect relationship or these parameters could be independent phenomena only related indirectly through the reduced metabolic activity of the quiescent cells.
Induction and decay of thermotolerance in rainbow trout fibroblasts
Authors: Mosser D. D. , van Oostrom J. , Bols N. C.
Published: February 2005

Thermotolerance was studied in the rainbow trout fibroblast cell line RTG‐2. RTG‐2 cultures that had been incubated at 28°C for 24 h were better able to withstand ultimately lethal temperatures above 28°C than RTG‐2 cultures that had been maintained at the routine growth temperature of 22°C. This thermotolerance developed rapidly between 3 and 6 h and was fully developed by 24 h at 28°C. After development for 24 hr at 28°C, thermotolerance showed little change over 72 h at 0 and 5°C but approximately a 40 and 60% reduction at 10 and 22°C, respectively. This is the first demonstration of heat‐induced thermal resistance in the cells of a poikilothermic vertebrate.
The glioma cell‐derived neurite promoting activity protein is functionally and immunologically related to human protease nexin‐I
Authors: Knauer Daniel J. , Orlando Robert A. , Rosenblatt Dorrie
Published: February 2005

Protease nexin‐I (PN‐I, Mr ∼ 43,000) is representative of a newly described class of cell‐secreted protease inhibitors. PN‐I has been purified to apparent homogeneity, partially sequenced, and monospecific antibodies have been raised against it. PN‐I is a potent inhibitor of urokinase, thombin, plasmin, and trypsin. In addition, cells have specific receptors that mediate the uptake of covalently linked complexes formed between PN‐I and its protease substrates. In the present studies, we have investigated the relationship between human PN‐I and a protease inhibitor derived from C6 glioma cells in culture that has neurite‐promoting activity. On the basis of co‐purification on heparin‐Sepharose, identical molecular weight, antibody cross‐reactivity, and receptor cross‐reactivity, we conclude that PN‐I and the glioma‐cell‐derived inhibitor are equivalent molecules.
Elevation of a potassium current in differentiating human leukemic (HL‐60) cells
Authors: Wieland Steven J. , Chou Robin H. , Chen Thelma A.
Published: February 2005

Human promyelocytic leukemia (HL‐60) cells display a novel voltage‐dependent outward current under voltage clamp. This current is present at low levels in the proliferative state and in granulocytes derived from HL‐60 cells which were induced to differentiate with retinoic acid. It is elevated in macrophages derived from HL‐60 cells after exposure to phorbol‐12‐myristate‐13‐acetate (PMA). The current is carried primarily by K+, is blocked by Cs+ and by increased intracellular concentrations of Cl−. From a holding potential of −80 mV, significant activation required depolarization to +20 mV membrane potential. Activation was not influenced by intracellular Ca2+ (1–2 × 10−6M). These properties appear to differ significantly from the Ca2+‐activated K+ channel and the delayed rectifier. The increase of this voltage‐activated current in differentiation toward the macrophage, but not the granulocyte, suggests that this current is correlated specifically with macrophage differentiation.
Phorbol myristate acetate inhibits growth in S49 cells: Isolation of resistant variants
Authors: Leach Karen L. , Powers Elaine A. , Mayo Judy K. , Abraham Irene , Burnett Bonnie‐Ann , Groppi Vincent E.
Published: February 2005

We have used S49 mouse lymphoma cells to study phorbol ester effects on growth. Treatment of wild‐type (wt) cells with phorbol 12‐myristate 13‐acetate (PMA) results in growth arrest within 72 hr. We have selected variants that are resistant to PMA‐induced growth arrest, based on a selection in the presence of 10 nM PMA. We have characterized one of these variants, termed 21.1, in detail. The 21.1 and wt cells contain similar levels of protein kinase C (PKC) as determined by [3H]phorbol 12,13‐dibutyrate ([3H]PDBu) binding. Treatment of both wt and 21.1 cells with PMA results in translocation of PKC to the membrane, suggesting that the coupling between PKC and an immediate biological response is intact. PMA treatment leads to the phosphorylation of many similar proteins in wild‐type and 21.1 cells. However, in the 21.1 cells there is a prominent substrate of approximately 70 kilodaltons (kD) which is no longer phosphorylated after PMA treatment. In wild‐type cells ornithine decarboxylase (ODC) activity and mRNA levels are decreased within 1 hr of PMA treatment. Likewise, ODC levels are decreased in the 21.1 cells after exposure to PMA even though PMA only slightly modulates the growth of these cells. The 21.1 cells represent a unique line with a dominant phenotype in which ODC expression is uncoupled from the growth state of the cell. These cells may represent a good model system in which to examine the steps involved in phorbol ester growth regulation in S49 cells.
Erythropoiesis in murine long‐term bone‐marrow cell cultures: Dependence on erythropoietin and endogenous production of an erythropoietic stimulating activity
Authors: Oddos Thierry , Nacol‐Lizard Sarab , Blanchet Jean Paul
Published: February 2005

Erythropoiesis was obtained in murine long‐term bone‐marrow cell cultures (LTBMCs) in the presence of erythropoietin (Epo) when the medium was frequently renewed. The level of the erythropoietic differentiation was shown to be a function of the erythropoietin concentration. In response to Epo addition, an activity which stimulates CFU‐E proliferation in semisolid cultures of fresh bone marrow cells was detected in the LTBMC supernatants. These results suggest that another factor, whose synthesis may be under Epo control, participates in the stimulation of erythropoiesis in vitro.
Role of biosynthesis of cholesterol and isoprenoid derivatives in regulation of G1 progression and cell proliferation of 3T6 cells
Authors: Larsson Olle
Published: February 2005

The growth of 3T6‐cells was rapidly decreased, although not completely stopped, as a consequence of treatment of the cell cultures with serum‐free medium. By analyzing the cell cycle traverse it was found that the decrease in growth rate was attributable to a 6–8‐h delay in progression through the proximal part of G1. These effects of serum depletion on cell‐cycle traverse and cell proliferation were compared with simulataneous effects on the activity of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase and the biosynthesis of cholesterol and the two isoprenoid derivatives coenzyme Q and dolichol. The activity of HMG CoA reductase was unaffected during the first 8 h of serum depletion and depressed by approximately 50% during the next 16 h. In contrast, the rate of coenzyme Q and dolichol synthesis, as related to cholesterol synthesis, was substantially decreased, and within 4 h after shift to serum‐free medium both were reduced by 70–80%. As distinguished from dolichol synthesis, the synthesis of coenzyme Q was even decreased following exposures of the cell cultures to cholesterol‐poor serum. This indicates that the rate of coenzyme Q synthesis is dependent on the concentration of serum cholesterol, whereas rate of dolichol synthesis is stimulated by some other serum factor(s). In addition, supplementation with dolichol to serum‐depleted cells partially normalized G1 traverse and DNA synthesis, whereas cholesterol or coenzyme Q was ineffective. Taken together, the results suggest that a certain level of de novo synthesis of dolichol is required to maintain normal cell‐cycle traverse and growth of 3T6 cells.
Production of platelet‐derived growth factor by cultured Wilms' tumor cells and fetal kidney cells
Authors: Fraizer Gail E. , Bowen‐Pope Daniel F. , Vogel Arthur M.
Published: February 2005

We have cultured human Wilms' tumors (nephroblastomas) in vitro and examined their growth properties, tumorigenicity in nude mice, karyotypes, and production of a platelet‐derived growth factor (PDGF)‐like activity (PDGFc). The cultured Wilms' tumor cells do not exhibit transformed growth properties, senesce after 15–25 passages, and are not tumorigenic in nude mice. Two of the three Wilms' tumors examined had karyotypic abnormalities but none contained a deleted 11p region. The Wilms' tumor cells and control human fetal kidney cells produce significant amounts of PDGFc and express few PDGF receptors on the cell surface. Analysis of Wilms' tumor cell RNA using probes specific for PDGF A‐chain and B‐chain (c‐sis) detects A‐chain but not B‐chain transcripts in the tumor cells.
Mouse embryonic stem cells exhibit indefinite proliferative potential
Authors: Suda Yoko , Suzuki Misao , Ikawa Yoji , Aizawa Shinichi
Published: February 2005

The proliferative potential of embryonic stem cells was examined. In contrast to the current concept of the finite life‐span being the hallmark of normal cells, we have been able to maintain these embryonic stem cells in vitro up to about 250 cumulative doublings with no indication of “crisis” or transformation. These cells could be considered normal on the basis of: (1) their apparently normal diploid karyotype, (2) their ability to extensively colonize embryos without causing tumors and developmental anomalies, and (3) their ability to form normal gametes when differentiated into the germ‐line. These results suggest that embryonic stem cells prior to differentiation into germ and somatic cells are indeed immortal.
Size‐dependent hyaluronate degradation by cultured cells
Authors: McGuire Paul G. , Castellot John J. , Orkin Roslyn W.
Published: February 2005

Hyaluronate degradation was examined in cultures of vascular wall cells (bovine aortic endothelial cells, rat aortic smooth muscle cells) and in nonvascular cells (chick embryo fibroblasts). The three cell types examined all produced hyaluronidase activity in culture which had a strict acidic pH requirement for activity. This suggested that the enzyme was active only within an acidic intracellular compartment and therefore that hyaluronate degradation occurred at an intracellular site. This was supported by the observation that the presence of hyaluronidase activity alone was not sufficient to ensure degradation of extracellular hyaluronate. Rather, the key limiting factor in this process appeared to be hyaluronate internalization, and this was found to be hyaluronate size‐dependent and to a degree, cell‐specific. The relationship of these results to morphogenesis and tissue remodeling is discussed.
Expression of gamma‐interferon receptor in murine bone marrow‐derived macrophages associated with macrophage differentiation: Evidence of gamma‐interferon receptors in the regulation of macrophage proliferation
Authors: Chen Ben D.‐M. , Chou Ta‐Hsu , Ratanatharathorn Voravit
Published: February 2005

The effect of purified, recombinant murine gamma interferon (IFN‐γ) on the regulation of macrophage proliferation induced by colony‐stimulating factor 1 (CSF‐1) was investigated. Although both hemopoietic stem cells (GM‐CFC) and tissue‐derived peritoneal exudate macrophages (PEM) proliferated in response to CSF‐1, the more mature PEM were much more sensitive to an antiproliferative effect of IFN‐γ. The role of IFN‐γ receptor expression and its relationship to growth inhibition was examined. Bone marrow cells as a whole did not exhibit an appreciable amount of IFN‐γ receptor binding activity. Likewise, nonadherent (NA) cells derived from CSF‐1‐stimulated bone marrow cultures displayed low levels of IFN‐γ receptor binding activity. On the contrary, more mature adherent (AD) cells (monocytes/macrophages) from the same culture exhibited high levels of IFN‐γ receptor binding activity which continued to increase with culture time. The elevated IFN‐γ binding activity is due to an increase in total receptor number rather than the binding affinity as judged by Scatchard analysis. Similar to the relationship between PEM and GM‐CFC, more mature AD cells were also more susceptible to the inhibitory effect of IFN‐γ on CSF‐1‐induced proliferation than their less mature NA counterparts. The fact that the sensitivity to IFN‐γ correlated well with the expression of existing IFN‐γ receptors strongly suggests that the inhibitory effect is mediated through IFN‐γ receptors. This study shows that the expression of IFN‐γ receptors in mononuclear phagocytes may not only represent one of the phenotypic parameters acquired by the growing macrophages during the process of differentiation, but may play some role in controlling proliferation.
Induction of cytokeratin expression in human mesenchymal cells
Authors: Von Koskull Harriet , Virtanen Ismo
Published: February 2005

We studied the phenotypic features of some typical human mesenchymal cells, including decidual stromal cells and adult and fetal fibroblasts under different cell culture conditions by using antibodies to intermediate filament proteins and desmoplakins. In cell culture, the decidual stromal cells rapidly acquired typical fibroblastoid appearance with abundant arrays of vimentin filaments while the cytokeratin‐positive epithelial cells, occasionally found in typical epithelioid colonies, lacked vimentin positivity and showed desmoplakin positivity. Within a few days, many of the stromal cells started to present cytokeratin positivity when cultured either in Condimed® or in Chang® medium. The cytokeratin positivity was first detected in small, scattered cytoplasmic dotted fibrils or in perinuclear dotlike aggregates with fibrillar projections. Later, denser cytokeratin‐positive fibrillar arrays could also be seen in stromal cells, which lacked desmoplakin positivity as judged by two monoclonal antibodies. Decidual stromal cells were also cloned and in five out of ten clones some of the cells acquired a similar cytokeratin positivity when transferred into Chang® or Condimed® medium. Immunoblotting results indicated that cytokeratins 8, 18, and 19 can be found in these cultures. Similar cytokeratin positivity could also be seen in the same culture conditions in cultured fetal fibroblasts from skin, chorionic villi, and lung but not in young or adult skin fibroblast cultures. The present results suggest that decidual stromal cells as well as some embryonal mesenchymal cells can acquire epithelial differentiation in vitro as judged by the emergence of cytokeratin proteins. This ability appears to be lost in the corresponding adult cell. The results furthermore suggest that cytokeratin fibrils can be organized in the cytoplasm without an apparent organization center and that neither the appearance of desmoplakins nor the formation of cell‐to‐cell contacts are required for cytokeratin filament assembly.
Induction of alkaline phosphatase in primary cultures of epiphyseal growth plate chondrocytes by a serum‐derived factor
Authors: Ishikawa Yoshinori , Valhmu Wilmot B. , Wuthier Roy E.
Published: February 2005

Alkaline phosphatase (AP) activity in epiphyseal growth plate cartilage increases markedly during differentiation of the chondrocytes, and reaches high levels in the zone of hypertrophy where vascular penetration and provisional mineralization begin. A proteinaceous factor has been discovered in serum that stimulates the expression of AP in chicken growth plate chondrocytes when these cells are grown in serum‐free media. Sera from a variety of vertebrate species (goat, fetal bovine, horse, human, and chicken) all contained detectable levels of the inducing activity. The chondrocyte AP‐induction factor (CAP‐IF) from fetal bovine serum was precipitated with ammonium sulfate between 33% and 50% saturation, and purified by dye‐ligand affinity chromatography. The active fraction, which eluted from an Affi‐Gel Blue column between 0.10 and 0.15 M NaCl, was further resolved on a QMA anion exchange column. The most active and almost homogenous fraction contained primarily a 64.5 kDa protein; about 3 μg/ml medium induced 50% of the maximal level of AP induction. CAP‐IF is stable to heat (100°C for 3 min) and dithiothreitol (50mM) treatment, and is only mildly inactivated by 2 h treatment with trypsin. CAP‐IF caused no significant effect on cell division as measured by 3H‐thymidine uptake. Time‐course studies revealed that at least 18–24 h exposure of the chondrocytes to CAP‐IF is required to produce major increases in AP activity. Longer exposure time generally further increases the response. Cycloheximide almost completely blocked the increase in AP activity, indicating that de novo protein synthesis is required for induction.
Metabolic effects of interleukin 3 on 32D cl23 cells analyzed by NMR
Authors: Hasthorpe S. , Carver J. A. , Rees D. , Campbell I. D.
Published: February 2005

31P NMR of living 32D cl23 cells and 1H NMR of cell extracts were used to study the metabolic effects of interleukin 3 (IL3). When IL3 was removed from 32D cl23 for 9–10 hours 31P spectra showed a decrease in sugar phosphate, γATP/ADP, αATP/ADP/NAD, and βATP resonances which declined progressively over a time period of up to 16 hours. By comparison, ATP measurements using the luciferin/luciferase method resulted in the decline of ATP levels from 12 hours in the absence of IL3. At this time, viability of the cells was unaffected. For 1H NMR experiments cells were grown in the presence and absence of IL3 for 4 and 24 hours, after which acid cell extracts were prepared. These spectra revealed a four‐fold decrease in lactate 4 hours post‐IL3 removal. Alanine levels were unchanged but glycine was elevated 1.5‐fold whilst various other amino acids were elevated slightly. After 24 hours without IL3, only 22% of cells were viable which was reflected in a general decline of most resonance intensities. These findings suggest that IL3 exerts its effect primarily on glucose metabolism and has a delayed secondary effect on maintenance of ATP levels in the cell. We have demonstrated the applicability of high resolution 1H and 31P NMR to the study of cellular metabolism in hemopoietic cells.
Altered hormonal responses: Markers for embryonal and embryonic carcinoma stem cells and their differentiated derivatives
Authors: Liapi Charis , Gerbaud Pascale , Anderson Wayne B. , Brion Daniele Evain
Published: February 2005

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the membrane adenylate cyclase of different embryonal carcinoma cell lines with differentiation. Each undifferentiated embryonal carcinoma stem cell studied (F9, PCC4, PC13, P19) has an adenylate cyclase system predominantly activated by calcitonin. Of great interest is the fact that cAMP production is also enhanced specifically by calcitonin in an embryo‐derived stem cell line. Differentiation of the embryonal carcinoma stem cell population toward parietal endoderm results in a decrease in calcitonin activation with a concomitant appearance of sensitivity to parathyroid hormone. Differentiation toward visceral endoderm is characterized by a lack of response of the adenylate cyclase system to both calcitonin and parathyroid hormone. These results indicate that the changes noted in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.
Modulation of fibronectin, laminin, and cellular adhesion in the transformation and differentiation of murine AKR fibroblasts
Authors: Chakrabarty Subhas , Brattain Michael G. , Ochs Robert L. , Varani James
Published: February 2005

The functional relationship between membrane/cell surface expression of fibronectin and laminin and transformation/differentiation was examined in an AKR mouse fibroblastic cell model. This model consisted of the untransformed AKR‐2B cells, their chemically transformed counterpart (AKR‐MCA cells) and the chemically differentiated from of the AKR‐MCA cells. The transformed AKR‐MCA cells were found to express more surface laminin and less fibronectin than the untransformed AKR‐2B cells. The transformed AKR‐MCA cells were slower to attach and spread on both plastic and type IV collagen‐coated dishes in comparison of the AKR‐2B cells. However, a higher percentage of the AKR‐MCA cells ultimately attached and spread on the type IV collagen‐coated dishes. The induction of differentiation in the AKR‐MCA cells by N, N‐dimethylformamide (DMF) restored fibronection to the surface of the AKR‐MCA cells but reduced laminin expression only slightly. The DMF‐treated AKR‐MCA cells resembled the AKR‐2B cells in that they rapidly attached and spread on plastic dishes and dishes coated with type IV collagen. They also resembled the AKR‐ MCA cells in that a high proportion ultimately attached and spread on the collagen‐coated dishes.
Hormone‐sensitive adenylyl cyclase in preadipocytes cultured from adipose tissue: Comparison with 3T3‐L1 cells and adipocytes
Authors: Kirkland James L. , Piñeyro Marco A. , Lu Zhongding , Gregerman Robert I.
Published: February 2005

The adenylyl cyclase system of preadipocytes derived from the stromal vascular fraction of perirenal rat fat pads was characterized. Unlike mature adipocytes, preadipocyte adenylyl cyclase was only weakly stimulated by catecholamines and adrenocorticotrophic hormone, but was stimulated by guanine nucleotides. Parathyroid hormone and 2‐chloroadenosine also stimulated preadipocyte adenylyl cyclase. The adenylyl cyclase system of preadipocytes resembled that of undifferentiated 3T3‐L1 cells. However, agents which induced the differentiation of the 3T3‐L1 cell adenylyl cyclase system did not have asimilar effect on predipocytes. A medium (CDM6) which induced some differentiation of preadipocyte adenylyl cyclase was developed. The observations that the adenylyl cyclase system of preadipocytes and undifferentiated 3T3‐L1 cells are similar, that preadipocyte adenylyl cyclase can be induced to develop along lines similar to early differentiation of 3T3‐L1 cells, and that the adenylyl cyclase system of fully‐differentiated 3T3‐L1 cells has characteristics intermediate between predipocytes and adipocytes, suggest that the differentiation of predipocyte and 3T3‐L1 adenylyl cyclase in vitro mimics adipose adenylyl cyclase development in vivo. The increased catecholamine and ACTH stimulation, and reduced GTP and adenosine sensitivites of adipocytes compared to preadipocytes suggest that a number of genes affecting adenylyl cyclase‐associated regulatory and receptor proteins are coordinately repressed and derepressed during development.
Mitogenic stimulation of human breast cancer cells in a growth factor‐defined medium: Synergistic action of insulin and estrogen
Authors: van der Burg Bart , Rutteman Gerard R. , Blankenstein Marinus A. , de Laat Siegfried W. , van Zoelen Everardus J. J.
Published: February 2005

The cooperative action of 17β‐estradiol (E2) and polypeptide growth factors in stimulating proliferation of human breast cancer cells in vitro was investigated. To prevent background estrogenic stimulation, only phenol red‐free media were used. When cultured in media supplemented with steroid‐stripped serum in which all polypeptide growth factor activity had been chemically inactivated, MCF7 cells were unable to proliferate and became virtually quiescent. In the additional presence of insulin, epidermal growth factor (EGF), and E2, however, cells proliferated as rapidly as did cells cultured in media supplemented with fetal calf serum. Analysis by DNA flow cytometry showed that in the absence of external growth factors, MCF7 cells became arrested predominantly in the G1/G° phase of the cell cycle. Upon addition of insulin in combination with EGF and E2, however, cells reentered the cell cycle with a high degree of synchrony. When added alone, E2 induced only slight mitogenic effects under these growth factor‐defined conditions. In contrast, this steroid induced optimal proliferation in conventional steroid‐stripped serum, which in itself contained considerable mitogenic activity. Insulin (at 10 μg/ml) was the most potent stimulator of MCF7 cell proliferation under growth factor‐defined conditions, resulting in a more than sixfold increase in cell number after 96 hours. Other growth factors such as platelet‐derived growth factor (PDGF), transforming growth factor β (TGFβ), and EGF had little effect by themselves and only slightly influenced insulin‐induced proliferation. At suboptimal concentrations of insulin (10‐100 ng/ml), however, strong synergism was observed between E2 and insulin in inducing MCF7 proliferation. Using the CG5 cell line, a highly E2‐sensitive MCF7 variant, synergism with E2 was already observed at 1 ng/ml insulin. It is concluded that MCF7 cells require insulin (or insulin‐like growth factors) for proliferation. At suboptimal insulin concentrations, E2 acts synergistically with insulin, possibly by inducing autocrine production of polypeptide growth factors by these cells.
Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum‐free medium
Authors: Pillai Sreekumar , Bikle Daniel D. , Hincenbergs Mara , Elias Peter M.
Published: February 2005

Growth and differentiation of keratinocytes in a serum‐free medium (keratinocyte growth medium of KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low‐calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H‐thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3–5 layers) surmounted by 2–4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum‐free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation.
Interleukin‐1 regulation of hematopoietic growth factor production by human stromal fibroblasts
Authors: Yang Yu‐Chung , Tsai Shickwann , Wong Gordon G. , Clark Steven C.
Published: February 2005

The human stromal fibroblastoid cell strain designated ST‐1 represents a normal population of cells capable of supporting hematopoiesis in vitro. These cells constitutively elaborate hematopoietic growth factor activity into the medium and the level of production of this activity dramatically increases following stimulation of the cells with IL‐1. This enhanced production is due at least in part to increased expression of the genes for GM‐CSF, G‐CSF, and IL‐6, but not IL‐3. The IL‐1 treatment had little effect on the expression of M‐CSF, a factor made constitutively by the cells. These results are consistent with the model that hematopoiesis is regulated at least in part by constant short‐range interactions of humoral factors produced by stromal cells both with other types of stromal cells and with the hematopoietic progenitors.
Hyaluronate appears to be covalently linked to the cell surface
Authors: Green Shawn J. , Underhill Charles B.
Published: February 2005

The purpose of this study was to examine the nature of the linkage between cell‐surface hyaluronate and the plasma membrane. To accomplish this, rat fibrosarcoma cells were cultured in the presence of [3H]‐acetate to isotopically label the hyaluronate, and then fixed with glutaraldehyde, which cross‐links proteins but does not react directly with hyaluronate. The glutaraldehyde fixation stabilized the cells so that they could be manipulated in ways which would otherwise destroy cells. The fixed cells were then subjected to various treatments, and the amount of hyaluronate remaining on the cell surface was assayed via exhaustive digestion with Streptomyces hyaluronidase. Using this technique, we found that (1) cell‐surface hyaluronate was quite stable for extended periods of time even in the presence of a large excess of non‐labeled hyaluronate; (2) 4 M guanidine HCI and detergents did not extract a significant portion of cell‐surface hyaluronate; (3) solutions of varying ionic strength (0–1 M NaCI) had no effect on the retention of hyaluronate; (4) the cell coat was stable in the range of pH 4–11, but outside this range a significant amount of hyaluronate was released; and (5) treatment with proteases released cell‐surface hyaluronate. These results are consistent with the possibility that hyaluronate is covalently linked to a protein associated with the plasma membrane. Further support for this model came from experiments with the detergent Triton X‐114, which can be used to separate soluble proteins from hydrophobic proteins. When nonfixed rat fibrosarcoma cells were extracted with this detergent and then partitioned by centrifugation, approximately 30 times as much hyaluronate was present in the detergent fraction which contained the hydrophobic proteins, as compared to the extracts pretreated with trypsin prior to phase separation. Again, these results suggest that cell‐surface hyaluronate is directly linked to a hydrophobic core protein intercalated in the plasma membrane.
Activation and proliferation signals in murine macrophages: Stimulation of glucose uptake by hemopoietic growth factors and other agents
Authors: Hamilton John A. , Vairo Gino , Lingelbach Suzanne R.
Published: February 2005

Purified colony‐stimulating factor (CSF‐1) (or macrophage colony stimulating factor [M‐CSF]) stimulated the glucose uptake of murine bone marrow‐derived macrophages (BMM) and resident peritoneal macrophages (RPM) as measured by 3H‐2‐deoxyglucose (2‐DOG) uptake. Similar concentrations of CSF‐1 stimulated the 2‐DOG uptake and DNA synthesis in BMM. Other purified hemopoietic growth factors, granulocyte‐macrophage CSF (GM‐CSF) and interleukin‐3 (IL‐3) (or multi‐CSF), and the tumor promoter, 12‐O‐tetrade‐canoyl‐phorbol‐13‐acetate (TPA), even though differing in their mitogenic capabilities on BMM, were also stimulators of 2‐DOG uptake in BMM and RPM. The nonmitogenic agents, lipopolysaccharide (LPS) and concanavalin A (Con A), were also active. The inhibition by cytochalasin B and by high concentrations of D‐glucose suggest that the basal and stimulated 2‐DOG uptake occurred via a carrier‐facilitated D‐glucose transport system. The responses of the two macrophage populations to the hemopoietic growth factors and to the other agents were quite similar, suggesting that events that are important for the induction of DNA synthesis are not tightly coupled to the earlier rise in glucose uptake. For the BMM, the ability of a particular agent to stimulate glucose uptake did not parallel its ability to promote cell survival. However, stimulation of glucose uptake could still be a necessary but insufficient early macrophage response for cell survival and subsequent DNA synthesis.
Modulation of cyclic AMP levels and differentiation by adenosine analogs in mouse erythroleukemia cells
Authors: Sherman Matthew L. , Shafman Timothy D. , Kufe Donald W.
Published: February 2005

Friend virus‐transformed mouse erythroleukemia (MEL) cells can be induced to undergo erythroid differentiation by a variety of compounds, including dimethyl sulfoxide (DMSO) and the adenosine analog xylosyladenine. The present studies have monitored the effects of the stable adenosine receptor ligand N6‐phenylisopropyladenosine (PIA) on induction of MEL cell differentiation. PIA has been previously shown to stimulate adenylate cyclase activity in rat hepatic and mouse Leydig l‐10 cells as well as inhibit adenylate cyclase in adipocytes. In the present study, PIA was ineffective as an inducer of the differentiated MEL cell phenotype. However, the results demonstrate that PIA inhibits the induction of MEL cell differentiation by DMSO and xylosyladenine. The extent of this inhibition as determined by benzidine staining, induction of globin RNA, and loss of self‐renewal capacity was dependent on PIA concentration. The results also demonstrate that PIA induces a rapid and sustained increase in cyclic AMP (cAMP) levels. Furthermore, there was a highly significant correlation between cAMP levels and inhibition of xylosyl‐adenine‐induced differentiation (r = 0.962, P < 0.0005). This relationship is further supported by the demonstration that prostaglandins E1 and E2 increase MEL cell cAMP levels and inhibit induction of the differentiated MEL cell phenotype. Moreover, PIA inhibited induction of MEL cell differentiation by butyric acid, diazepam, hypoxanthine, and the aminonucleoside analog of puromycin. These results suggest that cAMP may act as a negative regulatory signal in the induction of MEL cell differentiation.
Normalization of epidermal growth factor receptor and transforming growth factor production in drug resistant variants derived from adenovirus transformed cells
Authors: Sircar S. , Weber J. M.
Published: February 2005

Variants (G2, G5) resistant to the cancer chemotherapeutic drug methylglyoxal bis (guanylhydrazone) (MGBG) were isolated from adenovirus type 2 transformed rat brain cells (F4; Sircar et al., 1987). Although at least one of these variants continued to express the adenovirus Ela and Elb transforming proteins, they both exhibited a detransformed phenotype as witnessed by flat morphology, loss of anchorage independent growth, and tumor forming capacity. Reverse transformation suggested the possibility of changes in growth factor receptors and the production of transforming growth factors. To test this possibility, we investigated the status of epidermal growth factor receptors (EGF‐r) and transforming growth factor alpha (TGF‐α) production in F4, G2 and G5 cells. The level of 125I‐labeled EGF binding to intact drug resistant cells increased by 2‐ to 3‐fold compared to the transformed parental cell. Scatchard analysis suggests that increased binding was the result of increased receptor levels rather than altered affinity of receptor for ligand. The production of growth factors which compete with 125I‐labeled EGF binding declined in the detransformed G2 and G5 cells to a level intermediate between transformed (F4) and normal cells (FR3T3). EGF‐receptor increase and the complementary decrease in growth factor production in the drug resistant variants may be associated with detransformation.
Tumor necrosis factor stimulates proliferation of human osteosarcoma cells and accumulation of c‐myc messenger RNA
Authors: Kirstein Martina , Baglioni Corrado
Published: February 2005

The objective of this study was to establish whether human recombinant tumor necrosis factor (TNF) can significantly stimulate the proliferation of some tumor cells. Treatment with TNF had little or no effect on the growth of human tumor cells and murine NIH/3T3 cells cultured in medium with high serum concentration. Two tumor lines, SK‐MEL‐109 melanoma and HOS osteosarcoma cells, were adapted to grow in medium supplemented with 0.5% serum. The growth of these SK‐MEL‐109 cells was inhibited by TNF, but that of the HOS cells was greatly stimulated by TNF in a dose‐dependent way. Treatment with 10 ng/ml of TNF resulted in a two‐fold increase in the rate of cell division. This effect of TNF was also shown by measuring DNA and protein synthesis. The continuous presence of TNF was not required for its mitogenic activity on HOS cells cultured with 0.5% serum, since treatment for only one day with TNF resulted in prolonged growth stimulation. The failure of TNF to promote division of cells cultured in medium with 10% serum may possibly be explained by the presence of saturating amounts of growth factors in serum. Interferons abolished the mitogenic activity of TNF on HOS cells. Furthermore, TNF did not show synergism with insulin or epidermal growth factor in stimulating growth of these cells. The level of c‐myc mRNA was increased five‐fold after 30 minutes of treatment with TNF. This shows that TNF is a growth factor for HOS cells and that it induces accumulation of c‐myc mRNA.
Inhibition of imidazole‐induced tyrosinase activity by estradiol and estriol in cultured B 16/C3 melanoma cells
Authors: Kline Ellis L. , Smith Terry J. , Carland Karen A. , Blackmon Barbara
Published: February 2005

The effect of estrogens on tyrosinase (EC 1.14.18.1) activity was studied in B16/C3 melanoma cultures. Estradiol, estriol, and other related steroids failed to influence tyrosinase activity when added to the medium of proliferating cultures. Imidazole (10 mM), on the other hand, induced the activity of that enzyme 3‐fold, as reported previously. Estradiol and estriol blocked imidazole induction, however, unlike the other estrogenic compounds. The blockade occurred within 15 min of hormone addition and was reversible. Dose‐response studies revealed that the maximal estradiol effect occurred at 0.75 nM and the half‐maximal effect occurred at 0.5 nM. Estriol was more potent, with the maximal blockade occurring at ∼ 0.5 nM and half‐maximal effect at 0.25 nM. The induction of tyrosinase by imidazole and the blockade of this induction by estradiol and estriol could not be demonstrated in broken cell preparations, suggesting that direct enzyme activation‐inactivation was not involved. Studies utilizing inhibitors of protein and RNA synthesis suggest that this effect is mediated at a pre‐translational level and is independent of mRNA destabilization.
Effect of nicotine on transferrin binding and iron uptake by cultured rat placenta
Authors: McArdle Harry J. , Tysoe Jenny
Published: February 2005

The effect of nicotine on transferrin and iron transport in placental cells has been studied. Nicotine inhibits iron uptake but has little effect on the steady‐state levels of transferrin. The effect is temperature and concentration dependent and is not reversible. At a concentration of 15 mM nicotine inhibited transferrin endocytosis by 40%, while iron uptake was decreased by nearly 60%. Nicotine exerted a similar effect on reticulocytes, but other amines, either tertiary or quaternary, had little or no effect on either iron uptake or steady‐state intracellular transferrin levels. The results suggest that nicotine acts by blocking uptake, probably by acting as a weak base inhibiting iron release from transferrin, and inhibiting exocytosis with a resultant block of endocytosis. The concentrations required to exert an effect are too high to implicate inhibition of iron transport in the effects of smoking on pregnancy.
Enhanced transbilayer mobility of phospholipids in malaria‐infected monkey erythrocytes: A spin‐label study
Authors: Beaumelle Bruno D. , Vial Henri J. , Bienvenüe Alain
Published: February 2005

Using a recently described technique of electron spin resonance spectroscopy, we determined the rate of transbilayer mobility (flip) of the four major simian‐erythrocyte phospholipids in the erythrocyte membrane after infection by Plasmodium knowlesi. The development of the malarial parasite induces a very large increase in the flip rate of these phospholipids (mainly during the ring stage and at the beginning of trophozoite maturation). The half flip time fell from 2 and 3 hr in the case of choline phospholipids in healthy erythrocytes to less than 15 min in erythrocytes infected with the last stage of the parasite.