bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple fractions of sodium exchange in human lymphocytes
Authors: Negendank William , Shaller Calvin
Published: February 2005

Human lymphocytes contain a large, saturable fraction of K+ that exchanges slowly with K+ in the external medium, and a small non‐saturable fraction that exchanges rapidly. We determined whether or not Na+ exchanges in a similar manner with external Na+. Cells were pre‐equilibrated to ensure absence of net ion movements. Efflux was studied by loading with 22Na and transferring without washing to a non‐labeled medium. Influx was studied by transferring to labeled medium and separating large samples of cells at 6,000g. There are fast, intermediate, and slow fractions of Na+ exchange, with half‐times of 2, 14, and 120 minutes. At normal external K+, most cell Na+ exchanges rapidly, while at lower external K+ the Na+ that replaces cell K+ exchanges slowly. Parallel sources of fast and slow fractions, such as extracellular ones and subpopulations of cells, were ruled out by simultaneous 42K and 22Na fluxes and by a quantitative analysis of the combined K+ and Na+ content and flux data over a range of external K+ and Na+ levels. Five possible models of ion fluxes occurring in series were considered. Surface matrix, surface binding sites, and cytoplasmic channels with rapid nuclea exchange were eliminated as sources of the fast fractions. Therefore, the fast fractions of K+ and Na+ must reflect the permeability of the surface membrane. This left only two possible sources of the slow fractions. One, a subcellular compartment (e.g., nucleus), was eliminated by the combined content and flux data. We conclude that the slow fractions of ion flux are rate‐limited by adsorption onto and desorption from cellular macromolecules. The data support the association‐induction hypothesis and are understood by reference to two fundamental concepts: that of rapid solute exclusion from cell water existing in a polarized state; and that of solute accumulation limited by adsorption onto fixed anionic sites within the cell.
The cytoplasmic appearance of three functions expressed during the G0→G1→S transition is nucleus‐dependent
Authors: Jonak Gerald J. , Baserga Renato
Published: February 2005

tsAF8, ts13, tsHJ‐4, and TK−ts13 cells are G1‐specific temperature‐sensitive (ts) mutants of BHK cells that do not enter S phase when serumstimulated from quiescence at nonpermissive temperature (39.6°‐40.6°). TK−ts13 are, in addition, defective in thymidine kinase. Different G1 functions must be involved in these cells, since the first three cell lines complement each other when forming heterokaryons.

We have used these cells to study the role of the nucleus in the cytoplasmic expression of these G1 functions during the transition of cells from the non‐proliferating to the proliferating state. We fused cytoplasts from either serumstarved (G0) or serum‐stimulated (S) tsAF8 cells with G0‐ts13, G0‐tsHJ‐4, and G0‐TK−ts13 recipient cells and determined, after serum stimulation of the fusion products, which type of cytoplasts could complement the defective G1 functions. Cytoplasts from S‐tsAF8 cells complemented all three functions, i.e., cybridoids between S phase cytoplasts and ts13 or tsHJ‐4 recipient cells entered S at the nonpermissive temperature, and TK−ts13 recipient cells incorporated exogenous thymidine. Cytoplasts isolated from G0‐tsAF8 cells (3 days of serum starvation) complemented ts13 cells but not tsHJ‐4 and TK−ts13 cells. Cytoplasts from 6‐day starved tsAF8 cells lost the complementing capacity for ts13 cells. However, when the 6‐day starved tsAF8 cells were fused with G0‐ts13 cells, the heterokaryons entered S phase at the nonpermissive temperature. Also, cytoplasts isolated from the 6‐day starved cells that were serum stimulated for 40 hr before enucleation regained the capacity to complement ts13 cells.

These results demonstrate that three functions required in G1 cannot be detected in the cytoplasm of serum‐starved cells, although they are present in the cytoplasm of S‐phase cells. These results suggest that a functional nucleus is required for the cytoplasmic appearance of certain G1 functions in serumstimulated cells.
Growth of functional primary cultures of kidney epithelial cells in defined medium
Authors: Taub Mary , Sato Gordon
Published: February 2005

Primary cultures of baby mouse kidney epithelial cells can grow without fibroblast overgrowth in a hormone‐supplemented serum‐free medium (Medium K‐1) designed for an established kidney epithelial cell line, MDCK. The five supplements in Medium K‐1 are insulin, transferrin, PGE1, T3, and hydrocortisone. Medium K‐1 also supports the growth of kidney epithelial cell cultures from a number of animls, including man, withoug fibroblast overgrowth. Outgrowth of kidney epithelial cells from kidney explants was also observed with Medium K‐1. Thus, the medium appears to be selective for epithelial cell growth.

The physiological properties of primary cultures of baby mouse kidney epithelial cells were studied in detail. Baby mouse kidney epithelial cells grew at equal rates (0.5 doublings/day) in Medium K‐1 and serum‐supplemented medium. Medium K‐1 also supported the formation of baby mouse kidney epithelial colonies at low cell densities. The dependence of baby mouse kidney epithelial colony formation upon the five factors in Medium K‐1 was examined. These studies indicated that the formation of baby mouse kidney epithelial colonies in defined medium depended upon all the five supplements in Medium K‐1, in a manner similar, although not identical, to MDCK colonies.

Primary cultures of baby mouse kidney epithelial cells grown in Medium K‐1 retained kidney cell‐associated properties, including the ability to form multicellular domes, a phenomenon associated with transepithelial salt transport. Amiloride‐sensitive Na+ uptake and the mucosal surface enzyme leucine aminopeptidase were also observed in baby mouse kidney cultures. Similar functions were observed in MDCK monolayers.
Complement‐dependent induction of DNA synthesis and proliferation of human diploid fibroblasts
Authors: Korotzer Terry I. , Clagett James A. , Kolb William P. , Page Roy C.
Published: February 2005

The effects of fresh human serum (FHS) and heat‐inactivated human serum (HHS) on the DNA synthesis and proliferation of human diploid fibroblasts were assessed. FHS activated significantly more quiescent fibroblasts to undergo DNA synthesis and proliferation than did HHS. The stimulatory effect occurred consistently over a serum concentration range of 0.1–10%. Using bromodeoxyuridine selective killing techniques, it was shown that this FHS stimulatory effect was on a specific subpopulation of fibroblasts unresponsive to HHS. The involvement of the complement system, and specifically of C1, was shown by the inability of Clq‐depleted FHS to support enhanced DNA synthesis whereas Clq‐depleted serum reconstituted with purified Clq was effective. Purified Clq did not restore activity when added to heated serum, nor was it mitogenic when tested in basal medium without serum. The addition of purified Clq to fresh serum inhibited the enhancement of DNA synthesis, and at Clq concentrations of 4γ/ml and greater, the fresh serum effects were abrogated. Thus, it appears that binding of the assembled C1 complex to the fibroblast surface was required for FHS‐mediated enhancement of fibroblast proliferation, with Clq subcomponent serving as the recognition site. The results from several experiments indicated that antibody was not required for the complement‐dependent fibroblast activation. FHS was not cytotoxic, and autologous serum was as effective as allogeneic sera. A 20‐fold molar excess of Fab' from pooled human IgG did not alter the FHS effects. FHS from which IgG was more than 99% depleted was still effective. These results suggested an antibody‐independent role for complement in the activation of a subpopulation of human diploid fibroblasts.
Transmembrane potential changes associated with superoxide release from human granulocytes
Authors: Jones G. S. , VanDyke K. , Castranova V.
Published: February 2005

Treatment of human granulocytes with concanavalin A, phorbol myristate acetate (PMA), N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), and A23187 (a calcium ionophore) stimulates the release of superoxide anion and the generation of chemiluminescence. The fluorescent probe, Di‐S‐C3(5), has been used to monitor shifts in membrane potential in response to these stimulants which precede the secretion of superoxide. Concanavalin A, PMA, and FMLP induce a biphasic shift in transmembrane potential (Em), i.e., a rapid depolarization followed by a prolonged hyperpolarization. This depolarization is dependent on both external sodium and calcium while the hyperpolarization is inhibited by ouabain which blocks the electrogenic Na‐K pump. In contrast, A23187 induces a rapid and prolonged depolarization. This monophasic shift in Em is dependent on external calcium. These results suggest that depolarization acts as a signal to initiate events associated with the “respiratory burst” of these phagocytes.
Trifluorothymidine resistance and colony size in L5178y/TK+/− cells treated with methyl methanesulfonate
Authors: Amacher David E. , Paillet Simone C.
Published: February 2005

L5178Y/TK+/‐ cells treated with methyl methanesulfonate (MMS) were allowed to recover for 0,48,96,144, or 240 hours, and were then plated in soft‐agar medium containing trifluorothymidine (TFT). Dose‐dependent and consistent increases in the frequency of TFTR cells were observed after each of the 48–240‐hour expression periods through the counting of predominantly large, mutant colonies. Size distributions of soft‐agar colonies from either MMS‐treated or control cells were bimodal in the presence, and unimodal in the absence, of TFT. An increase of small, presumptive TFTR colonies with either increasing MMS concentration or decreasing recovery time was probably a manifestation of chemical toxicity, for a similar increase in small‐colony number was observed in the absence of TFT when cells were cloned immediately after MMS treatment, when no induced mutants were yet detectable. Recloning experiments with 22 small‐colony‐derived cell lines revealed that, with one exception, small‐colony morphology was not a heritable trait. While all large‐ and some small‐colony‐derived stocks from MMS‐treated cells were of the phenotypically stable TK−/‐ type; spontaneous small TFTR colonies generally were not, their occurrence being directly correlated with serum concentration. No aneuploidy was evident in MMS‐treated cell lines several generations after isolation as small TFTR colonies. These results suggest that delayed MMS cytotoxicity in TK+/‐ cells can temporarily produce increased physiological resistance to TFT in some cells, giving rise to secondary populations of small‐colony TFTR variants.
On the activity and fidelity of chromatin‐associated hepatic DNA polymerase‐β in aging murine species of different life spans
Authors: Fry Michael , Loeb Lawrence A. , Martin George M.
Published: February 2005

Activity and accuracy of chromatin‐directed DNA replication have been compared in young and aged Mus musculus and Peromyscus leucopus, two murine species with contrasting maximum lifespans. Chromatin isolated from livers of mature adults of both species copied efficiently exogenous DNA templates using predominantly DNA polymerase‐β. The DNA synthetic activity of liver chromatin remained constant in both species throughout their lifetimes. The fidelity of chromatin‐directed poly [d(A‐T)] synthesis was similar for the comparatively short‐lived M. musculus and the relatively long‐lived P. leucopus and remained unaltered in old animals. The fidelity of poly [d(A‐T)] copying catalyzed by DNA polymerase‐β dissociated from liver chromatin was comparable to that of the chromatin‐directed synthesis. The dissociated enzymes did not exhibit diminished fidelity of poly [d(A‐T)] synthesis with age. In all ages of both species examined, the murine liver DNA polymerase‐β, both chromatin‐associated and solubilized, exhibited high error frequencies; approximately one dGMP was incorporated for every 500–1,000 complementary nucleotides polymerized. The relationship of these results to the accuracy of DNA replication and repair as a determinant of aging is considered.
Mechanism of growth promotion of mouse lymphoma L1210 cells in vitro by feeder layer or 2‐mercaptoethanol
Authors: Ishii Tetsuro , Hishinuma Ieharu , Bannai Shiro , Sugita Yoshiki
Published: February 2005

Mouse lymphoma L1210 cells require some thiol compounds (such as 2‐mercaptoethanol) or feeder layer cells for their growth in normal culture media in vitro. We found that feeder layer cells (human diploid fibroblast IMR‐90) constantly produce thiol compounds; cysteine was the major thiol compound accumulating in the culture medium. In the culture medium of L1210 cells, added cysteine was rapidly oxidized and was toxic to the cells at high concentrations. However, cysteine promoted growth of L1210 cells when it was added repeatedly to the medium at low concentrations. These results show that the major role of feeder layer cells is to provide cysteine continuously. The glutathione content of L1210 cells depended largely on the cysteine concentration in the medium. In normal culture media containing cystine but not cysteine, the cellular glutathione content decreased notably within a few hours. Cysteine had to be supplied repeatedly to keep the content high. Cystine promoted the cellular glutathione content at unphysiologically high concentrations. These results were attributable to the extremely low uptake rate of cystine by the cells as compared with that of cysteine. In the presence of 2‐mercaptoethanol, the uptake of radioactive cystine by the cells was increased and a high cellular glutathione level was maintained. A thiol‐independent variant of L1210 took up cystine far more rapidly than L1210. From these results we concluded that the deficiency of the cystine uptake limits the growth of L1210 cells in normal culture media.
Multihormonal regulation of ornithine decarboxylase activity in highly differentiated porcine granulosa cells in vitro
Authors: Veldhuis Johannes D. , Sweinberg Sharon
Published: February 2005

Purified luteinizing hormone, but not follicle‐stimulating hormone, elicited time‐ and dose‐dependent stimulation of the cytosolic enzyme, ornithine decarboxylase, in highly differentiated, porcine granulosa cells maintained in vitro in chemically defined medium. Enzymic induction was susceptible to inhibitors of protein and RNA synthesis, and was suppressed by selective direct and indirect inhibitors of ornithine decarboxylase. Physiologic concentrations of prostaglandin E2 and L‐epinephrine also enhanced enzymic activity in a dose‐dependent and saturable manner. Systematic comparison of the hormonal induction of ornithine decarboxylase in highly differentiated versus poorly differentiated granulosa cells revealed distinctive patterns of enzymic responsivity in relation to the degree of cytodifferentiation attained in vivo.

This in vitro model is likely to permit further detailed examination of the molecular mechanisms subserving the hormonal control of ovarian ornithine decarboxylase activity in spontaneously differentiated granulosa cells maintained under chemically defined conditions in vitro.
Glutamine and the regulation of DNA replication and cell multiplication in fibroblasts
Authors: Zetterberg Anders , Engström Wilhelm
Published: February 2005

Several studies indicate that glutamine is a critical requirement for cell growth in vitro. Growing and quiescent (serum‐starved) 3T3‐fibroblasts were exposed to media (Dulbecco's modified Eagle's minimal essential medium) in which the concentration of the 13 essential amino acids had been lowered to 1/100 or 1/1,000 of that in DMEM ‐ either all together or one by one. The effects on DNA synthesis were measured by autoradiographic determinations of the percentage of labeled cells after 24 hours exposure to 3H‐thymidine. A reduction of all 13 essential amino acids to 1/100 or 1/1,000 of the normal concentration in the medium resulted only in a minor growth inhibitory effect during the first cell cycle. A similar growth inhibitory effect was caused by the depletion of one of the 13 essential amino acids (except glutamine) from the medium. However, a depletion of glutamine from the medium resulted in a marked inhibition of growth. Conversely, a relative excess of glutamine, when the other 12 amino acids were lowered to 1/1,000 of the normal concentration, counteracted the growth inhibitory effect of serum starvation. It was even possible to stimulate quiescent cells to undergo DNA synthesis by exposing them to a serum‐depleted (0.5% serum) medium with a relative excess of glutamine.
Properties of a sarcoma‐growth‐factor‐like peptide from cells transformed by a temperature‐sensitive sarcoma virus
Authors: De Larco Joseph E. , Preston Yvette A. , Todaro George J.
Published: February 2005

Serum‐free conditioned media was collected from three sarcoma virus‐transformed cell lines and an untransformed cell line. All three virally transformed lines produced and released growth factors into their serum‐free media. The major activity in all cases, whether the cells were transformed by Moloney sarcoma virus (MSV) or Kirsten sarcoma virus (KiSV), or whether they were mouse or rat, was a sarcoma‐growth‐factor (SGF)‐like activity with an apparent molecular weight of 10,000. The SGF‐like pools from a Moloney sarcoma virus‐transformed mouse 3T3 cell and a Kirsten sarcoma virus‐transformed NRK cell were further purified by carboxymethyl cellulose chromatography. The elution profiles of these peptides were very similar. The serum‐free conditioned media from the untransformed cells showed no detectable growth stimulating activity. The temperature sensitivity of an SGF‐like growth factor from the supernate of a NRK cell transformed by a wild‐type Kirsten sarcoma virus (KiSV) was compared with that of the SGF‐like activity from the supernates of a NRK cell transformed by a ts‐mutant of KiSV that is temperature sensitive with respect to transformation (ts‐371 Cl 5). Neither the cells transformed by the wild‐type sarcoma virus nor those transformed by the temperature sensitive virus released a SGF‐like activity that was temperature sensitive under the conditions of the assays.
Stretch‐induced growth of skeletal myotubes correlates with activation of the sodium pump
Authors: Vandenburgh Herman H. , Kaufman Seymour
Published: February 2005

Skeletal myotubes responded to passive stretch by increased amino acid uptake (as measured with [3H]α‐aminoisobutyric acid), increased incorporation of amino acids into total cellular protein and myosin heavy chains, and increased accumulation of total cellular protein and myosin heavy chains. These alterations were preceded by an increase in the uptake of ouabain‐sensitive rubidium‐86 (86Rb+), a potassium tracer used to measure membrane sodium pump activity (Na+K+ATPase). This stretch‐induced stimulation of 86Rb+ uptake resulted from a 60‐70% increase in the Vmax of the Na pump with little change in the Km. [3H] ouabain binding studies showed no stretch‐induced change in the number of membrane Na pumps, indicating that stretch activates the Na pumps that are already present on the cell surface. Since the stretch‐induced increases in amino acid transport and amino acid incorporation into proteins were inhibited by ouabain, Na pump activation may be involved in stretch‐induced cell growth of skeletal muscle cells by hypertrophy.
Glycolipids: Receptors for fibronectin?
Authors: Yamada Kenneth M. , Kennedy Dorothy W. , Grotendorst Gary R. , Momoi Takashi
Published: February 2005

We have examined the hypothesis that glycolipids might serve as receptors for the cell surface glycoprotein fibronectin using three different biological assay systems. We find that purified solubilized gangliosides inhibit fibronectin‐mediated hemagglutination, cell spreading, and restoration of a normal morphologic phenotype to transformed cells. The inhibition is dose‐dependent and competitive; hemagglutination by 2 μg/ml fibronectin is half‐maximally inhibited by less than 1 μM gangliosides. The most effective ganglioside inhibitors generally contain the most sialic acid residues. The isolated oligosaccharide portions of gangliosides retain this inhibitory activity and the oligosaccharides with more sialic acid are more effective inhibitors.

A series of other lipids or ganglioside constituents are either less effective or without detectable activity. The more active of these lipids are the more negatively charged phospholipids such as phosphatidyl serine and phosphatidyl inositol.

Our results support the hypothesis that the “receptors” for fibronectin on the cell surface either consist of or contain gangliosides or other negatively charged lipids.
Functional receptors for epidermal growth factor on human osteosarcoma cells
Authors: Shupnik Margaret A. , Tashjian Armen H.
Published: February 2005

Previous studies have shown that epidermal growth factor (EGF) stimulates bone resorption in organ culture via a prostaglandin‐mediated pathway, and that there are specific receptors for EGF on mouse bone (Tashjian and Levine, ′78; Shupnik et al., ′80). The present study demonstrates that a clonal line of human osteosarcoma cells, G‐292 (Clone A141B1) has specific, high‐affinity receptors for EGF and responds to treatment with EGF by increased prostaglandin production. Binding of 125I‐EGF to G‐292 cells exhibited a prolonged plateau phase (6 hours); thereafter, binding slowly declined (t1/2 = 6 hours) to 30–40% of the maximal level. This decrease in cell‐associated 125I‐EGF was prevented by leupeptin (50 μg/ml). EGF binding was of high affinity (Kd = 1.9 × 10−9 M) and was to a single class of non‐interacting binding sites. Pretreatment of cells for 48 hours with EGF caused a maximum threefold increase in PGE2 production, with a half‐maximal response at 9.8 × 10−10 M EGF. Increased PGE2 production was detectable within 2 hours and the constant presence of EGF was needed to maintain the response. Although EGF is mitogenic in several other systems, it did not increase DNA synthesis in the osteosarcoma cells. EGF treatment also did not increase medium or intracellular cyclic AMP in these cells, although parathyroid hormone and exogenous PGE2 (200 ng/ml) increased cyclic AMP three‐ to tenfold over control levels. Pretreatment with EGF decreased the level of subsequent 125I‐EGF binding; receptor number decreased to 30–40% of control after 48 hours of treatment, and the half‐maximal effect occurred with pretreatment concentrations of 1.6 × 10−9 M EGF. In all respects tested, the binding and biological actions of EGF on the human osteosarcoma cells were the same as those in whole mouse bone. G‐292 cells thus provide a convenient model system to study EGF action on osseous tissue.
Cell cycle‐specific glucocorticoid growth regulation of a human cell line (NHIK 3025)
Authors: Bakke Oddmund , Eik‐Nes Kristen B.
Published: February 2005

It has been reported that the human cell line NHIK 3025 has a specific cytoplasmic glucocorticoid receptor. When these cells were exposed to glucocorticoids, the cell cycle time was prolonged. Cells, synchronized by mitotic selection, were subjected to the synthetic glucocorticoid dexamethasone throughout the cell cycle. Only cells exposed in the first half of G1 phase had a lengthened cell cycle time. Most of the prolongation was also located within the G1 phase. The dexamethasone growth inhibition was reversible and could be detected only in the cell cycle where the cells were exposed to the steroid. DNA‐histograms of asynchronous cells were recorded by flowcytometry at various times after steroid exposure. These histograms also showed G1 phase sensitivity and G1 phase prolongation after exposure to dexamethasone. Our results thus indicate that these cells have a dexamethasone‐sensitive restriction point in mid‐G1 phase of the cell cycle.
Growth of human foreskin fibroblasts in a serum‐free, defined medium without platelet‐derived growth factor
Authors: Weinstein R. , Hoover G. A. , Majure J. , van der Spek J. , Stemerman M. B. , Maciag T.
Published: February 2005

The hormones which support growth, in vitro, of normal, neonatal human foreskin fibroblasts were determined. Wheresas thrombin and hydrocortisone were major growth stimulants, platelet‐derived growth factor was not. Human foreskin fibroblasts grew in a serum‐free, biochemically defined medium consisting of epidermal growth factor (100 ng/ml), insulin (100 ng/ml), trasferrin (10 μg/ml), thrombin (1 μg/ml), ascorbic acid (10 μg/ml), and hydrocortisone (5 × 10−5M) in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F‐12, supplemented with ovalbumin (1 mg/ml) and trace elements. The growth achieved was comparable to that achieved with 5% fetal bovine serum. Neither platelet‐derived growth factor, fibroblst growth factor, nor somatomedin activity increased proliferation. This serum‐free medium designated Defined Medium F, provides a biochemically defined system for growth and limited subcultivation of human foreskin fibroblasts in vitro.
Ability of endothelial cells to condition culture medium
Authors: Gajdusek Corinne M. , Schwartz Stephen M.
Published: February 2005

Endothelial cell‐conditioned medium contains two classes of factors distinguishable by behavior during dialysis and on specificity for cell type. One species, which diffuses through dialysis tubing with an exclusion limit of 6,000 to 8,000 daltons, supports growth of bovine aortic endothelial (BAE) cells in medium containing a growth limiting concentration of serum (0.2% serum). The production of this material appears to depend upon the presence of serum in the medium being conditioned. The activity increases with time of exposure of BAE cells to serum and with increasing concentration of serum present in the incubation medium. This activity cannot be replaced by exogenous epidermal growth factor, fibroblast growth factor, platelet‐derived growth factor, insulin, or thymidine. The second species, the endothelial cell‐derived growth factor (EC‐DGF), is retained by dialysis tubing with an exclusion limit of 6,000 to 8,000 daltons. ECDGF stimulates the growth of smooth muscle cells but does not support BAE cell growth in limiting serum concentrations. Unlike the dialyzable species, the production of ECDGF is independent of previous incubation of BAE cell cultures in serum. These studies suggest that BAE cells are able to utilize serum components to produce conditioning factors for their own growth that are distinct from the higher molecular weight ECDGF.
Morphological transformation of cells induced by Kirsten sarcoma virus transforming factor is independent of serine protease
Authors: Fulton R. Jerrold , Kaplan Paul L. , Hart David A. , Ozanne Brad
Published: February 2005

Reports from several laboratories have suggested that the virus transformed state may be maintained either by ectopically produced growth factors of alternatively by ectopically produced serine proteases including plasminogen activator. Here we show that the maintenance of transformation induced by Kirsten sarcoma virus induced growth factor(s) is independent of serine proteases in that (1) the factors are not themselves serine proteases, and (2) the growth factors do not induce the expression of detectable serine proteases or plasminogen activator.
Receptor‐associated changes of the catecholamine‐sensitive adenylate cyclase in glioma cells doubly transformed with Moloney sarcoma virus
Authors: Higashida Haruhiro , Miki Naomsa , Tanaka Tatsuya , Kato Kanefusa , Nakano Tamotsu , Nagatsu Toshiharu , Kano‐Tanaka Kyoko
Published: February 2005

A doubly transformed rat glioma cell line, designated C6V‐1, was obtained from rat glioma C6 cells by infection with a rat‐adapted variant of Moloney sarcoma virus (MSV‐M‐os). The C6V‐1 cells show karyotypic changes in chromosome number (43) and structure, while C6 cells possess a normal male karyotype. C6V‐1 and C6 cells were employed for characterization of a receptor‐adenylate cyclase system of the surface membrane. C6V‐1 cells showed lower adenylate cyclase activity than that of C6 cells, though the apparent Km for ATP in both types of cells was the same. The maximal stimulation of adenylate cyclase by isoproterenol was significantly reduced, and Kact for isoproterenol was approximately 18‐fold lower in C6V‐1 cells. When the concentration of β‐adrenergic receptors was measured by various concentration of [3H] dihydroalprenolol (DHA), the maximal binding sites of C6 and C6V‐1 cells were 760 and 230 fmol/mg protein, respectively, without any changes in the association constant for DHA. The concentration of isoproterenol required for 50% displacement of the [3H] DHA binding (Kd) was the same (around 1.5 × 10−6M) in both cells, measured in the presence of GTP. Thus the 19‐fold drop in the Kd/Kact ratio in C6V‐1 cells suggests an incomplete coupling of β‐receptors to adenylate cyclase. Cyclic AMP phosphodiesterase activity and cAMP content in C6V‐1 were lower than in C6 cells. Mitochondrial monoamine oxidase and cytosomal enolase activities, however, were somewhat higher in C6V‐1 cells. The results indicate that a set of changes in the receptors and in the cyclic AMP system of C6V‐1 cells. The results indicate taht a set of changes in the receptors and in teh cyclic AMP system of C6V‐1 is one of the specific alteations by transformation, even though those may not be the cause of cell transformation.
Transformation‐dependent loss of the hyaluronate‐containing coats of cultured cells
Authors: Underhill Charles B. , Toole Bryan P.
Published: February 2005

The sizes of hyaluronate‐containing coats on the surfaces of parent and virus‐transformed cell lines (3T3 vs. SV‐3T3; BHK vs. PY‐BHK) were compared according to the method of Clarris and Fraser (1968, Exp. Cell Res., 49: 181–193) in which fixed red blood cells were allowed to settle slowly on the surface of culture dishes containing the cells. The coats were seen as areas devoid of red blood cells surrounding each of the cultured cells and could be destroyed by the addition of small amounts of streptomyces hyaluronidase, an enzyme specific for hyaluronate. In the case of the parent cell lines (3T3 and BHK), the coats were clearly visible, whereas for their virus‐transformed counterparts (SV‐3T3 and PY‐BHK), the coats were either greatly reduced or absent. To confirm these observations, the amount of hyaluronate associated with each of the cell lines was measured using a direct chemical assay and shown to be significantly greater for the parent cell lines than for their virus‐transformed counterparts. In addition, the parent cell lines secreted greater amounts of hyaluronate into the medium and retained a larger fraction of the total amount of hyaluronate at the cell surface than the virus‐transformed cells. Thus the larger amount of hyaluronate on the surfaces of the parent cell types may be the result of both a faster rate of production and a decreased rate of release.
Regulation of dihydrofolate reductase gene transcription in methotrexate‐resistant mouse fibroblasts
Authors: Wu Jin‐Shyun Ruth , Johnson Lee F.
Published: February 2005

We have used a methotrexate‐resistant mouse 3T6 cell line (M50L3) that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of about 300 to study the regulation of DHFR hnRNA synthesis. We have previously shown that when resting (G0) M50L3 cells are serum stimuled to reenter the cell cycle, the amount and rate of synthesis of DHER and the content of DHER mRNA all begin to increase as the cells enter the S phase of the cell cycle. The increase in DHFR mRNA content is due to an increase in the rate of mRNA production. In the present study, we have used the technique of DNA‐excess filter hybridization to determine the rate of synthesis of DHFR hnRNA relative to total hnRNA at various times following serum stimulation. We found that the relative rate of DHFR hnRNA synthesis began to increase at about the same time (6 hours), and increased to about the same extent (three to fourfold by 15 hours following stimulation) as we observed previously for DHFR mRNA production. This suggests that the increase in DHFR mRNA production (and consequently DHFR gene expression) is controlled primarily, if not exclusively, at the level of transcription. We also studied the effect of addition of high concentrations of dibutyryl cAMP and theophyllne on DHFR gene transciption. We found that addition of these drugs at the time of stimulation completely blocked the increase in DHFR hnRNA synthesis as well as entry into S phase. Addition of the drugs at either 13 or 20 hours following stimulation led to a rapid decrease in DHFR hnRNA synthesis. The drugs were found to have little effect on the ability of the cells to complete S phase when they were added at 13 hours following stimulation. Our results suggest that high intracellular concentrations of cAMP may effect DHFR gene expression not only by preventing the progession of cells through the G1 phase of the cell cycle but also by affecting the rate of DHFR gene transcription in a more direct manner.
In vitro proliferation and lifespan of human diploid fibroblasts in serum‐free BSA‐containing medium
Authors: Kan Mikio , Yamane Isao
Published: February 2005

We have developed two serum‐free chemically defined media (RITC 78‐6 and RITC 80‐7) that support the growth in culture of human diploid fibroblasts to the same extent as Eagle's basal medium (BME) supplemented with 10% fetal bovine serum (FBS). These two media contain modified Eagle's minimum essential medium (MEM) supplemented with nonessential amino acids, various trace metals, organic compounds and growth factors [insulin, mouse epidermal growth factor (m‐EGF), transferrin and triiodothyronine (T3)]. RITC 80‐7 medium differs from RITC 78‐6 in that it contains thymidine, hypoxanthine, and vitamin B12 and supports the long‐term serial cultivation of human diploid cultures. The addition of commercial bovine serum albumin (BSA, 5 g/liter) to the medium enhances cell growth. This effect is not observed if BSA is first delipidized, but reconstitution of BSA with certain lipids restores its ability to promote growth. BSA has an inhibitory effect on cellular attachment but this is overcome when fibronectin (FN, 10 mg/liter) is added to the medium.
Increases in cyclic AMP potentiate competence formation in BALB/c‐3T3 cells
Authors: Wharton Walker , Leof Edward B. , Olashaw Nancy , Earp H. S. , Pledger W. J.
Published: February 2005

The ability of platelet‐derived growth factor and fibroblast growth factor to stimulate the initiation of DNA synthesis in quiescent BALB/c‐3T3 cells was enhanced by cholera toxin. However, the addition of cholera toxin to unsupplemented medium was not mitogenic, nor did cholera toxin increase the mitogenic potential of mediuum supplemented with platelet‐poor plasma. The enhancement of serum‐induced DNA synthesis by cholera toxin was due to a specific effect on competence formation and not plasma‐controlled progression. Cholera toxin increased the rate of competence formation during a transient exposure of quiescent cells to platelet‐derived growth factor; this rate was further increased by the addition of isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor. Intracellular cyclic AMP concentrations in quiescent BALB/c‐3T3 cells were increased 2‐ to 3‐fold after the addition of cholera toxin. The addition of cholera toxin plus 30 m̈M isobutylmethylxanthine caused an even greater (7‐ to 8‐fold) increase in the cellular levels of cyclic AMP. That these increases in cyclic AMP concentrations mediated at least part of the increased sensitivity of quiescent cells to competence factors was substantiated by the observation that 0.01 to 1 mM monobutrylcyclic AMP or 8‐bromocyclic AMP also caused a concentration‐dependent potentiation of competence formation in quiescent cells during a transient exposure to platelet‐derived growth factor.
Effects of trifluoperazine and mitogenic lectins on calcium ATPase activity and calcium transport by human lymphocyte plasma membrane vesicles
Authors: Lichtman Andrew H. , Segel George B. , Lichtman Marshall A.
Published: February 2005

The phenothiazine, trifluoperazine, and the mitogenic lectins, phytohemagglutinin (PHA) and Concanavalin A (Con A), were tested for their effects on human lymphocyte plasma membrane Ca‐activated Mg‐ATPase and ATP‐dependent calcium uptake. Trifluoperazine completely inhibited Ca‐uptake when present from the start of the assay at concentrations of 100 m̈M or more. When added during measurement of calcium uptake, trifluoperazine reduced the rate of vesicular calcium accumulation but was unlike the calcium ionophore, A23187, which caused a rapid release of accumulated calcium from the vesicles. Trifluoperazine also inhibited membrane vesicle Ca‐ATPase activity, but this inhibition was nonspecific since the Mg‐ATPase and Na, K‐ATPase activities were inhibited to similar extents at the same concentration of the phenothiazine. In contrast, concentrations of PHA and Con A, which are mitogenic for lymphocytes, did not cause any change in Ca‐uptake when added to suspensions of membrane vesicles. Con A had no effect and PHA had a weak inhibitory effect on Ca‐ATPase activity.
Inhibition of hepatoma cell growth by analogs of adenosine and cyclic AMP and the influence of enzymes in mammalian sera
Authors: Hargrove James L. , Granner Daryl K.
Published: February 2005

The following evidence suggests that inhibition of hepatoma cell (HTC) growth by cyclic nucleotides is an adenosine‐like effect that is greatly modified by the type and treatment of serum used in the culture medium and is probably not mediated by cyclic AMP‐dependent protein kinase: (1) Heating serum reduces its phospho‐diesterase content, thereby slowing metabolism of cyclic AMP and reducing the inhibition of HTC cell growth by cyclic AMP; (2) Using medium that contains phosphodiesterase but lacks adenosine deaminase causes adenosine to accumulate from cyclic AMP and increases the toxicity of cyclic AMP; 3) Uridine or cytidine reverse the growth inhibition caused by adenosine, 5'‐AMP or cyclic AMP; 4) adenosine, 5'‐AMP and N6 ‐(δ2‐isopentenyl) adenosine are more toxic for HTC cells than is cyclic AMP, and N6, O2‐dibutyryl cyclic AMP is not toxic; and 5) N6, O2'‐dibutyryl cyclic AMP inhibits growth of Reuber H35 cells, but uridine prevents this inhibition of growth. We conclude that most, if not all, of the inhibitory effects of cyclic AMP and N6, O2'‐dibutyryl cyclic AMP on HTC and Reuber H35 hepatoma cell growth are due to the generation of toxic metabolites.
Perturbation of the switch‐on of transcriptase activity in intermediate subviral particles from reovirus
Authors: Borsa J. , Sargent M. D. , Ewing D. D. , Einspenner M.
Published: February 2005

Intermediate subviral particles (ISVP) derived from reovirus represent a simple model system for the switch‐on of transcriptase function. In such particles the endogenous transcriptase is present in a switched‐off form, one step removed from the switched‐on state. Switch‐on of transcriptase function is an active process in this system and can be triggered by K+ ions. A variety of agents which affect gene expression in cells were tested for an effect on switch‐on in ISVP. Marked effects on switch‐on in ISVP were observed with a diverse group of test agents, including DMSO and other solvents, BUdR, TdR, caffeine, theophylline, and temperature. The correlation in response between ISVP and cells suggests that the ISVP system may be useful as a model for studying the biochemical mechanisms underlying the perturbative effects of such agents on gene expression in cells.
Response of articular chondrocytes to pituitary fibroblst growth factor (FGF)
Authors: Sachs Barton L. , Goldberg Victor M. , Moskowitz Roland W. , Malemud Charles J.
Published: February 2005

Rabbit chondrocytes from pooled articular joints have been delineated by their time of attachment of culture flasks after initiation of primary monolayer culture, either attached (48‐AT) or floating (48‐F) after 48 hours. A general population of chrondrocytes (attached after 72 hours, 72‐AT) was also studied. The growth‐promoting activity of pituitary fibroblast growth factor (FGF) and its effect on sulfated‐proteoglycan synthesis was studied on each chondrocyte population in secondary monolayer culture. 3H‐thymidine incorporation during a 1‐hour pulse was stimulated by FGF (100 ng/ml) in each chondrocyte population. The response of AT‐72 chondrocytes to FGF required an additional fetal bovine serum supplement, while 48‐F cells resonded independent of serum. The response of 48‐AT chondrocytes to FGF (100 ng/ml) during a 1‐hour pulse with 3H‐thymidine was increased in low serum (0.5–2.0%) rather than when high serum (8–10%) was present in the culture medium. FGF reduced 35SO4 incorporation into sulfated‐proteoglycans in the 48‐AT and 48‐F chondrocyte populations, but not in the 72‐AT population. The reduction in 35SO4 incorporation in the 48‐AT and 48‐F chondrocytes was not characterized by alterations in the hydrodynamic size of the sulfated‐proteoglycans as measured by Sepharose CL‐2B chromatography nor by changes in the types of sulfated‐glycosaminoglycans produced. These results indicated that FGF produced quantitative rather than qualitative alterations in chondrocyte sulfated‐proteoglycan synthesis. The latter appears uncoupled from the growth‐promoting activity of FGF on chondrocytes.
Production of monocyte/macrophage colony‐stimulating factor by preadipocyte cell lines derived from murine marrow stroma
Authors: Lanotte M. , Metcalf D. , Dexter T. M.
Published: February 2005

Three preadipocyte cell lines that have been independently derived from bone marrow stroma (Lanotte et al, 1982) have been tested for their capacity to produce granulocyte, macrophage, and erythroid colony‐stimulating factors (CSFs). All elaborated colony‐stimulating material that was active upon adult mouse marrow granulocyte/macrophage colony‐forming cells (M‐CFC) but not foetal liver GM‐CFC. The major activity was characterised as a monocyte‐macrophage colony‐stimulating factor (M‐CSF), and the pattern of colony stimulation was similar to that seen after addition of highly purified L‐cell CSF. Furthermore, the stimulating activity was specifically neutralised by rabbit anti‐L cell CSF antibodies. No evidence was found for stimulation of multipotential or erythroid colony‐forming cells, only few granulocytic colonies were detected, and the stimulating activity had no mouse strain restriction. All cell lines produced large quantities of M‐CSF; however, the production was found to be modulated during the adipogenesis process. A peak in M‐CSF production corresponded to the period of growth arrest after confluence of the stromal cells was reached and when adipocyte maturation was at an early stage. A marked depression in M‐CSF secretion was associated with the final steps of adipocyte maturation.
Isolation and characterization of peanut agglutinin‐resistant embryonal carcinoma cell‐surface variants
Authors: Rosenstraus Maurice J. , Hannis Margaret , Kupatt Laura J.
Published: February 2005

The isolation and characterization of variant embryonal carcinoma (EC) cells possessing altered cell‐surface structures is described. The lectin peanut agglutinin (PNA), which binds to EC cells but not their differentiated derivatives, was used to select the variants. Clones resistant to the cytotoxic effect of PNA were isolated at a frequency of 4 × 10–5 following mutagenesis. The resistant phenotype was stable in the absence of selection in all eight clones tested. The increased frequency of resistant clones following mutagenesis and the stability of the phenotype suggests a mutational origin. Somatic cell hybrids constructed between wild‐type cells and two different PNA‐resistant cell lines were sensitive to PNA; this suggests that the resistant phenotype is recessive. Binding assays demonstrated that resistant cells exhibited a twofold to fourfold reduction in the total amount of PNA bound. Together with the recessive behavior of the phenotype, this suggests that resistant cells are deficient for PNA receptors. The PNA‐resistant cells also showed reduced binding of monoclonal antibody against stage‐specific embryonic antigen 1 (SSEA–1) in indirect cytotoxicity tests. All eight PNA‐resistant lines isolated were tumorigenic in syngeneic mice and gave rise to well‐differentiated teratocarcinomas. The PNA‐resistant cells behaved like their wild‐type parents in a cell recognition assay; when incubated in suspension with endodermal cells, they sorted out to form simple embryoid bodies (a core of EC cells surrounded by an endodermal rind). Thus, EC cells can form tumors, differentiate, and recognize differentiated cells in a sorting assay despite a reduction in expression of the embryo‐specific cell surface structures (s) that bind PNA and anti‐SSEA‐1 antibody.
Attachment and growth of human keratinocytes in a serum‐free environment
Authors: Gilchrest Barbara A. , Calhoun Jean K. , Maciag Thomas
Published: February 2005

Using a serum‐free system, we have investigated the influence of human fibronectin (HFN) and selected growth factors (GF) on the attachment and growth of normal human keratinocytes in vitro. Single‐cell suspensions of keratinocytes from near‐confluent primary plates, plated on 5–10 μg/cm2 HFN, showed approximately 30–40% attachment after 2–24 hours of incubation at 37°C, compared with 4–6% attachment on uncoated platic plates. Percentage of attached cells was independent of seed density, tissue donor age, in vitro culture age, or medium composition, while subsequent cellular proliferation was strongly dependent on these factors. Keratinocytes grown on an adequate HFN matrix in a previously described hormone‐supplemented medium (Maciag et al., 1981a) achieved four to eight population doublings over 7–12 days at densities ≥ 104 cell/cm2. Removal of most GF individually from the medium had little or no effect on growth, while removal of epidermal growth factor (EGF) alone reduced growth by 30–35% and removal of bovine brain extract (BE) alone reduced growth by approximately 90%. Conversely, EGF alone in basal medium supported approximately 10% control growth, BE alone supported 30–40% control growth, and the combination of EGF and BE approximately 70%. In addition to its major effect on proliferation in this system, BE was necessary to preserve normal keratinocyte morphology and protein production. These findings expand earlier observations that HFN facilitates keratinocyte attachment in vitro and that a brain‐derived extract can exert a major positive influence on cultured keratinocytes.
Physicochemical and functional changes in human leukemic cell line HL‐60
Authors: Stendahl O. , Dahlgren C. , Hed J.
Published: February 2005

The recently established human promyelocytic cell line HL‐60 was induced to differentiate in the present of DMSO. During this process, physiochemical, and functional changes were detected simultaneously. After exposure to DMSO for more than 1 day, the cell volume decreased and the tendency for hydrophobic interaction increased. Using a hydrophobic two‐phase system in counter current distribution fashion, it was then possible to separate more mature metamyelocytes and segmented granulocytes from immature myeloblasts and promyelocytes. Increased functional maturity was reflected by increased chemiluminescence (CL) response and phagocytic activity. Using yeast particles opsonized with IgG as stimulating agent, the CL response increased already after 1 day in DMSO, in parallel with increased phagocytosis of these particles. In contrast, C3b‐opsonized yeast and phorbol 12‐myristate 13‐acetate (PMA) did not enhance the CL response conspiquously until days 3–4. These data suggest that Fc receptor function linked to phagocytosis and the activation of oxidative metabolism develop earlier than that of C3b and PMA. The dissociation between Fc‐ and PMA‐dependent stimulation of the oxidative metabolism may reflect different mechanisms of activation.