bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport of 6‐deoxy‐D‐glucose and D‐xylose by untransformed and SV40‐transformed 3T3 cells
Authors: Romano Antonio H. , Connell Nancy D.
Published: February 2005

Transport rates of the nonphosphorylated D‐glucose analogs 6‐deoxy‐D‐glucose and D‐xylose were measured in quiescent and serum‐stimulated cultures of mouse 3T3 cells, in SV40‐transformed 3T3 cells (SV101), and in a density revertant cell line derived from SV101 (Fl‐SV101). Initial rates of both entry and exit of 6‐deoxy‐D‐glucose and D‐xylose were more than threefold higher in serum‐stimulated 3T3 and in SV101 cells than they were in quiescent 3T3 cells, but transport rates were not higher in the transformed cells (SV101) than they were in serum‐stimulated 3T3. Confluent cultures of Fl‐SV101 showed lower rates of transport than serum‐stimulated Fl‐SV101, but not as low as quiescent 3T3 cells. These data confirm previous findings of others with other analogs that glucose transport is one of the cell functions that is depressed when 3T3 cells enter the quiescent G0 state, but emphasize that SV40‐transformed 3T3 cells do not show higher activity of the D‐glucose carrier than do actively growing 3T3 cells. Thus, enhanced glucose transport appears not to be a specific consequence of transformation, but a reflection of the active growth state of the cell.
Altered cell cycle progression and aberrant mitosis in adenovirus‐infected rodent cells
Authors: Murray James D. , Bellett Alan J. D. , Braithwaite Antony W. , Waldron Lydia K. , Taylor Ian W.
Published: February 2005

Actively growing mouse or rat embryo cells suffered structural chromosome damage, mitotic anomalies, and polyploidy after infection by human adenovirus type 5. Chromosome damage required expression of one or more early viral genes and showed regular periodicity in its frequency. The growth cycle time of some of the infected cells was reduced by about 5 hours due to a decrease in G1, and the interval between successive waves of chromosome damage corresponded to this reduced cycle time. After infection there was a decrease in cells with G1 DNA content and an increase in cells with G2 diploid, aneuploid, and polyploid DNA contents. We suggest these effects are due to the expression in semipermissive cells of early viral gene(s), whose function in productive infection in vivo is to alter cell cycle controls in order to maximize the number of cells able to replicate viral DNA and the time such cells spend in DNA replication.
Characterization of a postlavage, in situ pulmonary macrophage population
Authors: Drath David B. , Davies Paul , Shorey Jeannette M. , Gibran Nicole S. , Simpson Paul J. , Huber Gary L.
Published: February 2005

A postlavage in situ subpopulation of pulmonary macrophages (PM), biochemically distinct from the lavaged population, has recently been isolated from rats. After exhaustive bronchopulmonary lavage to extract the free lung cells, the lungs were excised, homogenized, and filtered, and the resultant cell suspension was allowed to form a monolayer on plastic Petri dishes. Electron microscopic morphometry failed to indicate any morphologic differences in the two populations. The postlavage in situ PM were more active metabolically during phagocytosis of zymosan particles or stimulation by phorbol myristate acetate (PMA) than the corresponding lavage population, as evidenced by greater superoxide generation. Macrophages prepared by either method became more avidly phagocytic when incubated with cell‐free medium isolated in the preparation of the in situ population. Peroxidase, an enzyme absent from the granules of PM separated by lavage techniques, was found in a granule‐rich fraction of the in situ macrophage. Catalase activity was found in similar amounts in both supernatants and granule‐rich fractions of both populations. The results support the concept of subpopulations of PM and suggest that these subpopulations are distinguished by their biochemical properties and their functional abilities.
Ornithine decarboxylase induction and the cytoskeleton in normal and transformed cells
Authors: Rumsby Gillian , Puck Theodore T.
Published: February 2005

ODC induction by fresh medium added to stationary, medium‐depleted, confluent cultures has been studied in transformed HeLa and CHO cells, and in normal human fibroblasts as an indicator of the resumption of cell multiplication. The transformed HeLa cell displays a more easily reversed G1 block, a higher peak ODC level, and a shorter time period for achievement of the peak ODC value than does the normal fibroblast. Low concentrations of microtubule depolymerizing agents like colchicine suppress ODC induction almost completely in the normal fibroblast, but hardly at all in the HeLa or CHO cells. Both transformed cells occasionally reveal a superinduction of ODC at very low colchicine levels (10−8‐10−7 M) and a more variable response to such agents than does the normal fibroblast. Higher concentrations of colchicine suppress ODC induction in all cells. Experiments with actinomycin D and cycloheximide indicate that the principal colchicine action involves inhibition at the level of protein or mRNA synthesis, rather than inactivation of the already synthesized enzyme. These experiments are provisionally interpreted as an indication that a microtubular system is needed to reinitiate certain steps associated with growth in G1‐blocked, normal cells, and that a second microtubular action terminating enzyme biosynthesis may exist. This microtubular control is defective in the transformed cells here studied. Specific microtubular actions necessary for initiation and termination of protein syntheses may occur throughout the cell reproductive cycle, and in the course of normal differentiation processes.
Effects of 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor
Authors: End David , Tolson Nathaniel , Yu Mei‐Ying , Guroff Gordon
Published: February 2005

The phorbol ester tumor promoter 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (125I‐EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent Ki of 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of 3H‐choline and 32P‐orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome‐associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of 125I‐EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.
Interactions of lectins with CHO cell surface membranes. I. Competition studies indicate concanavalin A and WGA bind to discrete populations of sites
Authors: Emerson D. , Juliano R. L.
Published: February 2005

The binding of radioiodinated lectins to the CHO cell surface was measured for the following affinity purified plant agglutinins; concanavalin A, wheat germ agglutinin, Ricinus agglutinins (I, II), pea agglutinin, peanut agglutinin, and Bandeiria simpliciafolia agglutinin (BSLI). The number of binding sites at saturation ranged from 6 × 105for BSL 1 to 5 × 107 for pea and Ricinus. Affinity constants calculated by the Steck‐Wallach procedure ranged from 2 × 105m−1L for pea lectin to 4.5 × 106 M−1 L for peanut lectin. Competition studies between homologous and heterologous radiolabeled and unlabeled lectins indicated that homologous unlabeled lectin could fully block binding of radiolabeled lectin. For heterologous pairs, a variety of results were observed. Of particular interest is the finding that concanavalin A and wheat germ agglutinin mutually failed to compete for binding indicating that these two lectins bind to distinct, nonoverlapping populations of surface sites. This finding suggests that the binding sites for WGA and Con A reside, for the most part, on discrete populations of membrane molecules; this concept is further validated in a companion paper in the upcoming issue of this journal (Schwartz et al., 1982).
Interactions of lectins with CHO cell surface membranes. II. Differential effects of local anesthetics on endocytosis of CON A and WGA binding sites
Authors: Schwarz M. A. , Harper P. A. , Juliano R. L.
Published: February 2005

Using fibroblastic CHO cells, we have examined (1) the internalization and redistribution of surface binding sites for the lectins Concanavalin A and wheat germ agglutinin and (2) the sensitivity of these processes to putative inhibitors of cytoskeletal activity. Following initial exposure to fluorescein conjugated Con A (CAF) or WGA (WGAF) at 4° C, kinetic analysis of internalization and intracellular aggregation of lectin at 37°C indicated more rapid aggregate formation in the case of WGA than in the case of Con A. Treatment with tertiary amine local anesthetics (tetracaine, dibucaine, and xylocaine) or with a lysosomatrophic amine, m‐dansyl cadaverine, blocked internalization of Con A but not of WGA. Similar differential effects on redistribution of Con A and WGA were not however observed with the antimicrotubule agents colchicine and nocodazole. Simultaneous treatment of cells with WGAF and with rhodamine labeled Con A (CAR) resulted in the accumulation of both labels in a central perinuclear aggregate; a similar experiment in the presence of local anesthetic resulted in a diffuse peripheral distribution of CAR and a central aggregate of WGAF. These results suggest (1) CHO cells possess at least two distinct pathways for lectin internalization and redistribution, which can be discriminated in terms of drug sensitivity; (2) CHO cells can sort out and independently internalize different populations and lectin binding sites; and (3) different pathways may be a manifestation of biochemically distinct linkages between cytoskeletal elements and various groups of surface glycoproteins. Present findings concur with our previous results concerning the mutual independence of the surface binding sites for Con A and WGA (Emerson and Juliano, 1982).
Relation between sodium‐coupled amino acid and sugar transport and sodium/potassium pump activity in isolated intestinal epithelial cells
Authors: Sepúlveda F. V , Burton K. A. , Brown P. D.
Published: February 2005

Cells isolated from the epithelium of the small intestine are used to study the relationship between amino acid or sugar‐coupled sodium transport and potassium uptake through the sodium/potassium pump. Potassium influx is a saturable function of the external potassium concentration. Uptake in the presence of ouabain, a specific pump inhibitor, is greatly reduced. This remaining influx is linearly related to the concentration up to 6 mM potassium. Sugars and amino acids are actively accumulated by the intestinal cells. Their transport is accompanied by an initial extra influx of sodium. Although cells seem to regulate their internal sodium concentrations, this is not accompanied with a concomitant increase in potassium uptake through the pump. Thus L‐alanine, 3‐0‐methyl‐D‐glucoside, and α‐methyl‐D‐glucoside all fall to increase the rate of ouabain‐sensitive potassium uptake. A very high coupling ratio of sodium efflux to potassium influx through the pump would be a likely explanation of the present results though they cannot be regarded as conclusive.
Enzymatic modification of the surface carbohydrates of friend erythroleukemic cells
Authors: Suyama Kimita , Goldstein Jack
Published: February 2005

Surface carbohydrates of Friend erythroleukemic‐cells were modified by treatment with the exoglycosidases, α‐galactosidase, β‐galactosidase, and neuraminidase without affecting cell growth and viability either in the presence or absence of 1.8% DMSO as inducer. When cells were incubated with a combination of α‐galactosidase and neuraminidase and then induced, they showed an increased rate of differentiation as measured by the formation of benzidine‐positive cells. These enzymes used singly, or β‐galactosidase treatment alone, or in combination with neuraminidase, did not change the rate of differentiation. Cell‐surface labeling and electrophoretic separation of the glycoconjugates revealed that two regions of approximate molecular weights of 195,000 and 185,000 were neuraminidase‐sensitive and one other of molecular weight of about 75,000 was sensitive to α‐ galactosidase. Both untreated and the combined α‐galactosidase, neuraminidase‐modified cells exhibited the same rate of uptake of carbon‐14 DMSO, ruling out the possibility that the observed increased rate of differentiation was due to faster penetration of DMSO into enzyme‐treated cells. On the other hand, the decrease in the rate of uptake of rubidium ‐ 86, an analogue of K+, by treated‐induced cells was significantly enhanced over that observed with untreated‐induced cells, suggesting that α‐galactosidase plus neuraminidase modification of the cell surface was affecting at least one of the early events occuring in the Friend erythroleukemic cell differentiation program.
Rna dependence in the cell cycle of V79 cells
Authors: Fujikawa‐Yamamoto Kohzaburo
Published: February 2005

The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.
Effects of serum fractions on the growth of mononuclear phagocytes
Authors: Yen Shing‐Erh , Stewart Carleton C.
Published: February 2005

The effects of serum fractions on the growth kinetics and colony formation of mononuclear phagocytes derived from mouse bone marrow, blood, and peritoneal cavity were investigated. Peritoneal exudate macrophages and blood monocytes required a factor(s) found to reside in the nondialyzable serum fraction (molecular weight > 12,000) to survive, a small molecular weight (< 307) factor(s) with growth‐stimulatory activity (GSA) contained in the dialyzable serum fraction, and the macrophage growth factor (MGF) for proliferation and colony formation. Fetuin, a major protein of fetal serum, was able to substitute the non‐dialyzable serum fraction. Macrophages cultured in medium containing MGF and the nondialyzable serum fraction for 6 days could be restored to full growth following the addition of the dialyzable serum fraction. In contrast, bone marrow mononuclear phagocytes cultured in the absence of the dialyzable serum fraction were capable of proliferating, though at a slower rate, and forming colonies. In addition, neither insulin nor hydrocortisone was capable of replacing the serum‐dialyzable GSA nor able to enhance colony formation.
Analysis of purified fetal liver hemopoietic progenitor cells in liquid culture
Authors: Nicola Nicos A. , Metcalf Donald
Published: February 2005

The action of different types of colony‐stimulating factors (CSFs) on purified fetal hemopoietic progenitor cells in liquid cultures was studied. Three types of CSF were examined–pregnant mouse uterus extract (PMUE), which contains macrophage CSF; mouse lung conditioned medium (MLCM), which contains granulocyte‐macrophage CSF; and pokeweed mitogen‐stimulated spleen cell‐conditioned medium (PWM‐SCM), which contains ganulocyte‐macrophage, eosinophil, megakaryocyte, and erythroid CSFs. Without a source of CSF, the cells died within 48 hr, but with CSF, cells entered an exponential growth phase within 18 hr with a doubling time of about 12 hr. Althogh growth was best with PWM‐SCM, this appeared to be due to increased numbers of surviving cells during the early period of culture and a decreased lag phase rather than a shortening of the doubling time. With PWM‐SCM, the absolute increase in cell number was 40‐fold. Over the culture period of seven days, the blast cells also differentiated morphologically to end cells, although differentiation appeared to be highly asynchronous. With MLCM and PMUE, only cells in the granulocyte‐macrophage series were seen, but with PWM‐SCM, erythroid cells were also observed. PMUE neither stimulated nor allowed the survival for longer than two days of granulocyte or erythroid committed progenitors (CFC) but, in contrast, stimulated a marked increase in macrophage CFC, which were uniquely responsive to PMUE stimulation. The proliferation of these progenitors appeared to be actively inhibited by PWM‐SCM. Despite the inability of MLCM to stimulate mature erythroid cell production, it was able to stimulate the early proliferation of erythroid CFC as well as granulocyte‐macrophage CFC. PWM‐SCM was able to stimulate the early and late proliferation of all types of CFC.
Stimulation of embryonic mouse limb bud mesenchymal cell growth by peptide growth factors
Authors: Kaplowitz Paul B. , D'ercole A. Joseph , Underwood Louis E.
Published: February 2005

The possible role of peptide growth factors in mammalian intrauterine cell growth has been investigated using primary cultures of undifferentiated mesenchymal cells from 11‐day mouse embryo limb buds. When grown as monolayer cultures, proliferation is greatly favored by high cell densities. In medium containing 0.2% serum, purified epidermal growth factor (EGF), fibroblast growth factor (FGF), multiplication stimulating activity (MSA), insulin, and somatomedin‐C (Sm‐C) do not increase cell growth, but a 30–40,000 molecular weight component of mouse fetal liver conditioned medium is stimulatory. On the other hand, when limb bud cells are grown as high density or micromass cultures, a method which better approximates in vivo growth conditions, all of the purified growth factors tested stimulate cell growth significantly. These growth factors have additive effects when used in combination, the best stimulation being observed with liver medium (10% v/v), EGF (10 ng/ml), FGF (200 ng/ml), and either insulin (1 m̈g/ml) or Sm‐C (20 ng/ml). We conclude that the response of limb bud cells to growth stimulation is influenced by the manner in which the cells are cultured and that at least four different growth factors are required for optimal in vitro proliferation. One of these, the active component of liver medium, appears to be a previously uncharacterized growth factor.
Cyclic AMP does not modulate hexose uptake of serum‐deprived BHK‐21 fibroblasts
Authors: Lavis Victor R. , Davenport Roxann , Pontier Paul , Thompson W. Joseph , Strada Samuel J.
Published: February 2005

We have studied the uptakes of the glucose analogs, 2‐deoxyglucose (2DG), and 3‐O‐methylglucose (3OMG) in serum‐deprived BHK‐21 fibroblasts. Incubation for 4 hours with 0.25 mM 1‐methyl‐3‐isobutylxanthine (MIX) increased cellular cyclic AMP 3 to 15 fold, without affecting the initial rates of uptake of these sugars. Incubation with 100 nM insulin, doubled hexose uptake without affecting cyclic AMP. Insulin did not modify the cyclic AMP response to MIX; nor did MIX alter the hexose uptake response to insulin. There was no effect of 0.1 mM L‐isoproterenol (ISO) or 0.1 mM prostaglandin E1 (PGE1) on cyclic AMP. PGE1 slightly stimulated uptake of 2DG but not 3OMG; ISO did not affect hexose uptake. We conclude that cyclic AMP does not directly regulate hexose uptake in these cells.
Reversible inhibition by human serum lipoproteins of cell proliferation
Authors: Ito Fumiaki , Takii Yuko , Suzuki Jiro , Masamune Yukito
Published: February 2005

Normal human serum or plasma was studied for the presence of inhibitors of cell proliferation by assaying inhibition of incorporation of labeled thymidine into acid‐insoluble fraction using human FL cells. Lipoprotein fraction obtained by gel filtration through Sepharose 4B and by KBr density gradient centrifugation was found to play a major part of the inhibitory activity of the serum. It was also shown that the inhibitory activity resides in low‐density lipoprotein (LDL). The addition of the lipoprotein fraction to growing FL cells caused an early decrease in the transport of uridine and thymidine across the membrane. This change in the permeability of membrane was followed by the preferential inhibition of DNA synthesis and a reduction in the percentage of mitotic cells in the cell population. The inhibition of the growth was reversible and was observed in various types of cells irrespective of species.
Species specific differences in the toxicity of mithramycin, chromomycin A3, and olivomycin towards cultured mammalian cells
Authors: Gupta Radhey S.
Published: February 2005

Three structurally related anticancer drugs, mithramycin, chromomycin A3, and olivomycin, showed large unexpected differences (up to more than 1000 fold) in their toxicity towards cultured cells from various species (human, Chinese hamster, Syrian hamster, and mouse). Among the cell types examined, human cells (both a diploid fibroblast cell strain and HeLa cells) were maximally sensitive to all these drugs, followed by the Syrian hamster kidney cells (BHK 21). The mouse (LMTK− cells) and Chinese hamster (CHO) cells, which were more resistant, showed interesting differences in their sensitivity towards these drugs. For example, whereas the mouse cells were more resistant to mithramycin than CHO cells, the sensitivity pattern was reversed for both chromomycin A3 and olivomycin. In cell extracts derived from human, mouse, and Chinese hamster cells RNA synthesis, which is the cellular target of these drugs, showed identical sensitivity to both mithramycin and chromomycin A3, indicating that the species specific differences in the toxicity to these drugs are at the level of cellular entry of these compounds. Based on the structures of these glycosidic antibiotics and their patterns of toxicity, it is suggested that the intracellular transport of these drugs involves specific interactions between the sugar residues on these compounds and some type of cell surface receptor(s), which differ among different cell types. Some implications of these results for toxicity studies are discussed.
The amount of a specific cellular protein (p53) is a correlate of differentiation in embryonal carcinoma cells
Authors: Chandrasekaran Krish , Mora Peter T. , Nagarajan Lalitha , Anderson Wayne B.
Published: February 2005

A specific cellular protein of molecular weight of 53–55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS‐2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS‐2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might acount for the diminished levels of this protein, the amount ofp53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8‐bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.
Self‐renewal and commitment to differentiation of human leukemic promyelocytic cells (HL‐60)
Authors: Fibach Eitan , Peled Tony , Rachmilewitz Eliezer A.
Published: February 2005

More than 80% of cells from a human promyelocytic leukemic cell line (HL‐60) possess the capacity for self‐renewal as evidenced by their ability to form large primary colonies in semisolid medium and the presence within these colonies of cells capable of subsequent colony formation. Colony development is independent of the normal regulator‐the myeloid colony stimulating factor. The observed autostimulation suggests the production of specific growth promoters by the cells. Differentiation either to mature granulocytes or macrophages, induced by various agents, was associated with reduced cloning potential. Nevertheless, colonies containing differentiated cells could be developed either by cloning cells in the presence of suboptimal concentrations of inducer or by adding inducers over colonies developed in its absence. Upon differentiation, there was a morphological change from compact to diffused colony morphology due to cell mobility in the semisolid medium. Even at suboptimal concentrations of inducer more than 95% of the colonies became diffused, indicating clonaI homogeneity of the population with respect to differentiation capacity. The loss of self‐renewal was found to be one of the early properties which changed following the initiation of differentiation. The loss preceded not only the overt expression of maturation‐specific functions but also cellular commitment to terminal differentiation; shorter contact with the inducer was required to cause loss of self‐renewal than to induce an irreversible transition to differentiation. This resulted in cells that lost their self‐renewal potential without being able to complete their program of differentiation.
Effect of exogenous gangliosides on human neural cell division
Authors: Icard‐Liepkalns Christine , Liepkalns Vis A. , Yates Allan J. , Rodriguez Zoe R. , Stephens Ralph E.
Published: February 2005

Human neural cells in exponential growth phase were transferred to a serum‐free medium and maintained for 72 hr without any detectable loss in viability. The two normal fetal cell lines (CHI and CHII) showed a serum‐dependent cell proliferation, but the glioblastoma multiforme cells (12–18) were able to continue proliferating in this totally synthetic medium. The incorporation of [3H] thymidine into the acid‐precipitable fraction of both normal and neoplastic human neural cells was assayed in the presence and the absence of exogenous gangliosides by a convenient new method. In serum‐free medium, gangliosides (50 μM) inhibited the thymidine incorporation into the normal fetal cells within 24 hr and, in serum containing medium, reduced their proliferation within 48 hr. No such effects were detectable in the glioma cells. The inhibition of thymidine incorporation in the normal cells was reversible upon removal of the gangliosides. These results indicate a role of gangliosides in the postmitotic phase of normal human neural cells resulting in the regulation of cell proliferation.
Control of cytolysis of BALB/c‐3T3 cells by platelet‐derived growth factor: A model system for analyzing cell death
Authors: Scher Charles D. , Young Shelley A. , Locatell Kathryn L.
Published: February 2005

In culture medium supplemented with 10% clotted blood serum, the saturation density of BALB/c‐3T3 cells is determined jointly by cell replication and cell loss. By prelabelling cellular DNA with 3H‐thymidine and also by time lapse photography, we studied cell loss independently of replication. Cell loss was accelerated when BALB/c‐3T3 cells were transferred from serum‐supplemented medium, which contains the platelet derived growth factor (PDGF), to medium supplemented with platelet‐poor plasma which lacks it. Loss occurred via the disintegration of cell attached to the surface of the tissue culture dish. Cytolysis of individual cells occurred rapidly; less than 15 minutes transpired between the first indication of a perturbance (by phase contrast microscopy) and fragmentation of the cell cytoplasm. Kinetic analysis was consistent with random cell death rather than a fixed lifetime.

The percentage of cells undergoing cytolysis was governed by the cell density; at high densities, such as are present in confluent cultures, a higher percentage of cell loss was noted than at low density. Cell death was antagonized by partially purified or electrophoretically homogenous preparations of‐PDGF. Pure PDGF stimulated cell survivial at ng/ml in a concentration dependent fashion. The process of cell replication was not necessary for survival because PDGF prevented cytolysis in the presence of methotrexate, an inhibitor of DNA synthesis. A brief (4 hour) treatment with PDGF prevented cell death; such PDGF treated cells displayed increased survival after being taken up with trypsin and planted onto a fresh surface in plasma supplemented medium. Pituitary fibroblast growth factor, a functional analogue of PDGF for induc of DNA synthesis in BALB/c‐3T3 cells, also functioned as an anticytolytic agent. By contrast, epidermal growth factor and insulin did not. Cytolysis of SV40‐transformed cells occurred at a constitutively low rate and was insensitive to PDGF.
The characteristics of an anchorage‐independent clonal agar assay for primary explanted bovine granulosa cells
Authors: Bertoncello I. , Bradley T. R. , Chamley W. A. , Hodgson G. S.
Published: February 2005

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage‐independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4‐fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells.

Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self‐renewal as assessed by the ability of cells from primary colonies to redone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl‐1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.
Human mutant cell lines with altered RNA polymerase II
Authors: Shander Monica T. M. , Croce Carlo , Weinmann Roberto
Published: February 2005

A human fibrosarcoma cell line, HT‐1080‐6TG‐9AM, resistant to α‐amanitin at concentrations up to 10 μg/ml, was isolated after ethylmethanesulfonate mutagenesis and stepwise selection. The mutation is stable and dominant. RNA polymerase II purified from the mutant cells showed an altered affinity for labeled α‐amanitin and the sensitivity of the enzyme to the fungal toxin was decreased 50‐to 100‐fold. This functional test demonstrated that the biochemical basis for the resistance of the cells to α‐amanitin is due to an alteration of RNA polymerase II.
Rapid induction of foci escaping density‐dependent inhibition in baby mouse skin cultures
Authors: Cooper Peter D. , Marshall Susan A. , Masinello Gillian R.
Published: February 2005

Mesenchymal cells from primary BMS (baby mouse skin) cultures formed secondary monolayers subject to density–dependent inhibition. The monolayers remained quiescent but in good condition for 6–8 weeks if given weekly medium changes. Exposure of the primary cultures to fluorescent light and/or oxygen produced “altered” cells that gave rise in the secondary cultures to foci of up to 10 4 cells after 15 days. These foci overgrew the background BMS cells. The rate of growth, morphology, and arrangement of the altered cells varied greatly between foci but much less within a focus, which usually showed one or more characters of neoplastic cells. The initiation of foci was apparently not transmissible by an infectious agent.
Properties of cell lines derived from altered‐cell foci in baby mouse skin cultures
Authors: Cooper Peter D. , Marshall Susan A. , Masinello Gillian R.
Published: February 2005

Long‐term cell lines were readily developed from a proportion of either baby mouse skin (BMS) cultures passing through colchicine‐induced crisis or altered‐cell foci selected from BMS cultures exposed to light and/or oxygen followed by colchicine. The developing cell lines behaved as though they passed through a continuing, profound genetic reshuffling process, which was usually lethal but which in some cases eventually yielded a gene set that favored long‐term survival. Some cell lines have passed 120 cell doublings, and none has shown a second crisis or signs of senescence. As soon after isolation as measurement was possible (19–50 days) the cell lines were predominantly or entirely tetraploid or subtetraploid. Although BMS cells and all of the cell lines were density inhibited, the BMS, C14, C21 and C23 cells overgrew (formed colonies on) monolayers of the same cells in most combinations. The cell lines retained a variety of neoplastic morphological characters, although their morphology was more normal than in the original focus. No cell line, however, showed anchorage‐independent growth or formed tumors in syngeneic hosts. The cell lines may all, therefore, be regarded as preneoplastic.
Growth and death of hela cells in the presence of caffeine
Authors: Beetham Karen L. , Tolmach L. J.
Published: February 2005

Proliferation and death were measured in synchronously growing cultures of HeLa S3 cells during treatment with up to 30 mM caffeine. Changes in the number of colony‐forming cells were determined by single‐cell plating, while changes in the total number of cells were measured both by electronic counting and by monitoring cell division and physiological death cinemicrographically. At concentrations between 2 and 5 mM, cell killing occurs over several days during which the cells traverse the generation cycle once or a few times before losing colony‐forming ability, with consequent proliferation of non‐colony‐forming cells. This indicates that lethal damage is accumulated with time. Death occurs more rapidly at higher concentrations, without proliferation, the kinetics of inactivation being strongly dependent on the phase of the cycle (cell age) at which treatment is initiated. G1 cells are killed more slowly in 10 mM caffeine than are S cells, but G1 cells respond rapidly to 20 mM caffeine, suggesting the inception of an additional mode of killing. The incidence of sister‐cell fusion increases with increasing caffeine concentration above 1 mM. On addition of 10 mM caffeine to a culture prepared from collected mitotic cells, the cells undergo a transient rounding and then respread after several hours; with 20 mM, they never respread. The generation cycle is prolonged in a concentration‐dependent fashion, as is the duration of G1; the generation time is doubled in 5–6 mM caffeine. G2 and M are also prolonged at concentrations above 3 mM, but S is not prolonged even by 10 mM caffeine.
The effects of temperature and ouabain on steady‐state Na and K exchanges in human lymphocytes
Authors: Negendank William , Shaller Calvin
Published: February 2005

The temperature‐dependence of steady‐state exchanges of K and Na were determined under conditions in which cell viability, ATP, water, Ca, and Mg were not confounding variables. Steady‐state ion contents are near‐normal between 37° and 10° C, Below 10° C K is replaced by Na in a mole‐for‐mole fashion with significant net K retention and Na exclusion occurring even below 3°C. The rates of steady‐state Kand Na exchanges have markedly different temperature‐dependences; between 37° and 10° C, for example, that of K decreases markedly while that of Na remains near‐normal, and there is no consistent correlation between the steady‐state exchanges and the ionic contents. Ouabain increases steady‐state Na flux at 37° C and induces a more marked temperature‐dependence over the entire temperature range. This effect is not due simply to inhibition of some processes and unmasking others; it mirrors a pronounced effect of ouabain on the intrinsic properties of Na self‐exchange. These results are compatible with a model based on two simple concepts: (1) partial ionic exclusion from cellular water that is ordered by interaction with proteins; and (2) ionic accumulation mediated by adsorption onto and desorption from fixed macromolecular anionic sites, the majority of which interact with one another in a cooperative fashion. In this view, the sharp temperature transition in the net replacement of K by Na below 10° C is due to a critical transition in the selectivity of the cooperatively interacting adsorption sites. The rates of steady‐state self‐exchanges of K and Na are determined by parameters of ion‐site interaction, and the major set of ion‐adsorbing sites that interact cooperatively have a steep thermal activation energy of ionic self‐exchange. When they are in the K‐preferring state above 10° C, exchange of K has a steep temperature‐dependence. When they are in the Na‐preferring state below 10° C, exchange of Na has a steep temperature‐dependence. When these sites are forced into a Na‐preferring state at all temperatures by treatment with ouabain, exchange of Na acquires a steep temperature‐dependence over the entire temperature range 37° to 0° C.
Cell heterogeneity during the cell cycle
Authors: Darzynkiewicz Z. , Crissman H. , Traganos F. , Steinkamp J.
Published: February 2005

Using flow cytometry, populations of Chinese hamster ovary cells, asynchronous and synchronized in the cycle, were measured with respect to cellular RNA‐ and protein‐content, as well as cell light scatter properties. Heterogeneities of cell populations were expressed as coefficients of variation (c.v.) in percent of the respective mean values. Populations of cells immediately after mitosis have about 15% higher c.v. than mitotic cell populations, regardless of whether RNA, proteins, or light scatter are measured. These data indicate that cytoplasmic constituents are unequally distributed into the daughter cells during cytokinesis and that unequal cytokinesis generates intercellular metabolic variability during the cycle. An additional increase in heterogeneity, although of smaller degree, occurs during G2 phase. Populations of S‐phase cells measured in the selective window equivalent to 15–60 min progression through the cycle, i.e., comparable with the mitotic and postmitotic populations, are the most uniform, having 20–30% lower c.v. than the postmitotic cells. Cell progression through S does not involve any significant increase in intercellular variability with respect to RNA or protein content. In unperturbed exponentially growing cultures a critical RNA content is required for G1 cells prior to their entrance into S. Thus, the cells equalize in G1 with respect to RNA and protein and, during the transition from the period (compartment) of equalization (G1A) to the prereplicative compartment (G1B), they exhibit minimal heterogeneity. The cell residence times in the equalization compartments are exponentially distributed, which may reflect the randomness generated by the uneven division of metabolic constituents to daughter cells during cytokinesis. The cell heterogeneities were presently estimated at two metabolic levels, transcription (RNA content) and translation (proteins). The most uniform were populations stained for RNA and the highest variability was observed after staining of proteins. This suggests that the regulatory mechanisms equalizing cells in the cell cycle may operate primarily at the level of DNA transcription.
Contribution of glucose and gluconeogenic substrates to insulin‐stimulated glycogen synthesis in cultured fetal hepatocytes
Authors: Plas C. , Forest N. , Pringault E. , Menuelle P.
Published: February 2005

The influence of medium composition on basal and insulin‐stimulated glycogenesis was studied in cultured 17‐day‐old rat fetal hepatocytes, which contain no glycogen at the time of transplantation. Continuous‐labeling 14C‐glucose experiments were used to determine both glycogen content and glycogen labeling. The specific activity of glucose units in the newly formed glycogen (a) was compared to that of the medium glucose (b): the ratio a/b expresses the contribution of medium glucose to glycogen formation. In standard medium (5.5 mM glucose), this ratio averaged 0.60. Variations of glucose concentration in the medium from 1 to 40 mM were accompanied by a progressive increase in both glycogen content and the ratio a/b (up to 0.80). Supplementation of standard medium with fructose, galactose, glycerol, or lactate‐pyruvate decreased the hepatocyte glucose uptake from the medium. Galactose (1 to 5 mM) or lactate‐pyruvate (5 mM) enhanced the glycogen content whereas glycerol or fructose (1 to 5 mM) had no effect. The ratio a/b, not modified by glycerol or lactate‐pyruvate, was decreased to 0.45 by fructose (5 mM). Galactose at concentrations as low as 1 to 2 mM brought the ratio down to 0.30, indicating that it is a superior precursor of glycogen as compared to glucose. When the hepatocytes were grown in the presence of 10 nM insulin, the glycogen content was constantly higher than in the absence of the hormone (2‐fold stimulation). Also the amplitude of the glycogenic effect of insulin was similar whatever the modifications of the medium, whereas ratio a/b and glucose uptake were hardly increased by insulin. Thus several substrates can contribute to glycogen formation (especially galactose) in cultured fetal hepatocytes and the essential effect of insulin is a stimulation of the final step of the glycogenosynthetic pathway.
Putrescine dependent growth of mycoplasma infected mammalian cells
Authors: Kamatani Naoyuki , Willis Erik H. , McGarrity Gerard J. , Carson Dennis A.
Published: February 2005

The aliphatic diamine putrescine, a metabolic precursor of the polyamines spermidine and spermine, markedly stimulated the growth of a murine lymphoblastoid cell line (R 1.1) infected with Mycoplasma orale, under conditions of arginine limitation. The diamine acted by suppressing the growth of the mycoplasma, which use arginine as a major energy source, and thereby prevented the depletion of arginine from the medium. The antimycoplasmal effects of putrescine occurred at concentrations that were neither stimulatory nor toxic to uninfected cells.
Virus replication in infected epithelial cells is coupled to cell shape‐responsive metabolic controls
Authors: Ben‐Ze'ev Avri
Published: February 2005

The cell shape of monkey epithelial cells was varied from flat to spheroidal by gradually reducing the substrate adhesiveness with poly (HEMA) films of increasing thickness. The decrease in cell spreading is accompanied by a dramatic response in cellular macromolecular metabolism in the nucleus. Within 14 to 16 hr, DNA and RNA syntheses are inhibited by more than 95%, while the level of protein synthesis is reduced by only twofold after 24 hr in spheroidal‐suspension culture. When epithelial cells, spread to various degrees, are infected with SV40 or herpes virus a parallel inhibition of virus replication and cellular macromolecular metabolism occurs. However, VSV can proliferate in the metabolically active cytoplasm of epithelial cells in which nuclear activity is inhibited owing to alterations in cell shape. The results suggest that the metabolic restrictions imposed on epithelial cells, owing to changes in cell spreading, are a dominant phenomenon that cannot be overcome by virus infection. Rather, virus replication, which is dependent on the cellular metabolic machinery, is inhibited in parallel with the inhibition of cellular macromolecular metabolism.
The growth requirements of BHK‐21 cells in serum‐free culture
Authors: Bradshaw Gary L , Sato Gordon H. , McClure Don B. , Dubes George R.
Published: February 2005

A serum‐free medium for serial culture of baby hamster kidney cell line 21 (BHK‐21) as container‐adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F‐12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty‐acid‐free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm2, this serum‐free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum‐free medium was poor unless it was supplemented with serum‐free medium which had been conditioned by BHK‐21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.
[125I]EGF binding ability is stable throughout the replicative life‐span of WI‐38 cells
Authors: Phillips Paul D. , Kuhnle Ellen , Cristofalo Vincent J.
Published: February 2005

Using a serum‐free medium supplemented with hormones and growth factors, which included epidermal growth factor (EGF), we investigated the binding and processing‐degradation of [125I]EGF in WI‐38 cells of various in vitro ages. The binding and processing‐degradation systems of these cells remained esentially unchanged throughout their lifespan. The number of specific [125I]EGF binding sites per cell increased as the cultures senesced, thought the number of specific binding sites per μm2(surface area) remained constant. The kinetics of ligand degradation as well as the qualitative and quantitative nature of the degradation products also remained essentially unchanged throughout the life‐span. The only consistent alteration in any of the binding parameters measured was the slight decrease in the apparent kd of the ligand‐receptor complex, independent of temperature. Quantitation of EGF‐stimulated DNA synthesis revealed a decrease in the percentage of cells incorporating [3H]thymidine ([3H]TdR) during a 30‐h exposure from 45% in young cells to 0.25% in senescent cells, although [125I]EGF binding or processing‐degradation did not differ significantly in yong and old cells. Thus, EGF binding does not decrease in senescence.