bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane effects of tumor promoters: Stimulation of sugar uptake in mammalian cell cultures
Authors: Lee Lih‐Syng , Weinstein I. Bernard
Published: February 2005

Phorbol esters stimulate 2‐deoxy‐D‐glucose (DG) uptake in rodent and human cell cultures. The potent tumor promoting agent, 12‐0‐tetradecanoyl phorbol‐13 acetate (TPA), induces a 12‐fold stimulation in confluent 3T3 cells and a 2.5‐fold stimulation in HeLa cells. When a series of macrocyclic diterpenes are assayed, their relative potencies in stimulating DG uptake in 3T3 cells correlate with other known biologic effects of these compounds. On a molar basis, TPA is a much more potent stimulator of DG transport than insulin or epidermal growth factor. In HeLa cells, the ED50 value of the TPA effect is 0.2 nM. The increase in DG uptake occurs immediately after the addition of TPA, reaches a maximum at 90 minutes, persists for at least three hours after removal of TPA from the medium, and is temperature dependent. The stimulation is not inhibited by cycloheximide or actinomycin D. As in control cells, DG uptake in TPA treated cells is inhibited by p‐hydroxymercuribenzoate, phloridzin, cytochalasin B, and dexamethasone. Although the precise mechanism is not known, evidence is presented that the TPA stimulation of DG uptake is due to enhanced transport of the sugar rather than to effects of intracellular metabolism. The enhanced transport may be secondary to a more generalized change in membrane structure.
Bromodeoxyuridine‐induced reversion of transformed characteristics in BHK21 cells: Changes at the plasma membrane level
Authors: Lage‐Davila A. , Krust B. , Hofmann‐Clerc F. , Torpier G. , Montagnier L.
Published: February 2005

When BHK21 cells transformed by hamster sarcoma virus aregrown in the presence of 5‐Bromodeoxyuridine (BUdr), several in vitro properties of the transformed cells such as morphology, adhesiveness, and alignment, revert towards a state close to that of untransformed cells. We have studied plasma membrane changes associated with this phenotypic reversion by several different biochemical methods. Reversion is accompanied by a reappearance of Fibronectin, an increase in a membrane‐associated protein of M. W. 195,000, a decrease in glycosylation and exposure of a glycoprotein M. W. 100,000 which is increased in transformed cells and a decrease in Con A‐agglutinability. On the other hand, several membrane changes associated with malignant transformation namely, the increase in an integral membrane protein M. W. 177,000, the higher rate of hexose uptake, the increase in high molecular weight surface glycopeptides and, to some extent, the increase in the density of intramembrnous particles, did not revert under BUdr treatment. Thus, membrane properties of transformed cells may be dissociated into two main groups by BUdr treatment. In addition, the exposure and glycosylation of a growth‐regulated membrane protein M. W. 160,000 was highly sensitive to BUdr.
The effect of homologous localanesthetics of the 4‐alkoxy‐ and 4‐alkylamino‐benzoicacid‐diethylamino‐ethylester‐hydrochloride‐series on the glucose transport in human erythrocytes
Authors: Lacko L. , Wittke B. , Lacko I.
Published: February 2005

The homologous compounds of the 4‐alkoxy‐ and 4‐alkylamino‐series inhibit the exchange transport of glucose in human erythrocytes; they show a competitive inhibition with one or two inhibitor molecules which become bound to a singular site of the transport system for glucose. The importance of length of hydrocarbon chain of the localanesthetics for the mode of their action is discussed.
Cell cycle analysis of sodium butyrate and hydroxyurea, inducers of ectopic hormone production in HeLa cells
Authors: Fallon Robert J. , Cox Rody P.
Published: February 2005

Sodium butyrate and hydroxyurea, effective inhibitors of DNA synthesis in HeLa cells, cause these cells to produce increased levels of the ectopic glycopeptide hormones human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), and free α chains for these hormones. The objective of this study was an assessment of the role of modulation of cell cycle events in the action of these two chemical agents. A variety of experimental approaches was employed to obtain a clear view of the drugs' effects on cells located initially in all phases of the cell cycle. Cells in early G1, G2, or M phase at time of addition of either inhibitor were not arrested at early time points, but by 48 hours became collected at a location characteristic for each drug, near the G1‐S phase boundary. Flow microfluorometry (FMF) and thymidine labeling index revealed that butyrate‐treated cells arrested late in G1 phase very close to S phase, while hydroxyurea‐blocked cells continued to early S phase. Both inhibitors prevented cells originally in S phase from reaching mitosis. S cells exposed to hydroxyurea were killed by 48 hours, but those growing in 5 mM butyrate progressed to the end of S or G2 phase where they became irreversibly arrested although not removed from the monolayer. Analysis of the cell cycle location and viability of each subpopulation resulting from 48 hour exposure to butyrate or hydroxyurea is important for the study of the function of each cellular subset. Treatment of HeLa cells with lower concentrations of butyrate (1 mM) resulted in slowed yet exponential growth. Fraction labeled mitosis (FLM) analysis shows that this is a result of prolongation of the G1 phase.
Hemicyst formation stimulated by cyclic AMP in dog kidney cell line MDCK
Authors: Valentich J. D. , Tchao R. , Leighton J.
Published: February 2005

Certain epithelial cell lines have morphologic, physiologic, biochemical and pharmacologic characteristics of transporting epithelia from intact organs. In this paper we show that dibutyryl cyclic AMP, 5′ AMP, adenosine and cyclic AMP phosphodiesterase inhibitors stimulate hemicyst formation by the dog kidney cell line MDCK. It is suggested that this effect is explained by elevation of intracellular cyclic AMP levels by means of an exogenous non‐metabolizable source of cyclic AMP, phosphodiesterase inhibition or adenyl cyclase stimulation. Since hemicyst formation is in part due to transepithelial fluid transport, these findings raise the possibility that this fraction might be modulated by cAMP in an established cell line. We believe that cultured epithelial cells may provide an exploitable model system to investigate at the cellular and subcellular levels, the mechanism by which cyclic AMP modifies water and solute movements across epithelia.
Presence of epidermal growth factor receptors and influence of epidermal growth factor on proliferation and aging in cultured smooth muscle cells
Authors: Bhargava Girija , Rifas Leonard , Makman Maynard H.
Published: February 2005

Epidermal growth factor (EGF) at nanomolar concentrations stimulated DNA synthesis in confluent, serum‐starved cultures of calf aorta and human uterine smooth muscle cells. Stimulation of DNA synthesis in lens epithelial cells was studied for comparison. L and D‐ascorbic acid potentiated the effect of serum and EGF on DNA synthesis in calf aorta cells. In contrast L‐ascorbic acid had minimal potentiating effect with serum and no effect with EFG present along with serum on DNA synthesis in human uterine smooth muscle and rabbit lens epithelial cells. EGF and ascorbic acid increased cell number when added to stationary phase cultures. Specific binding of 125I‐labelled EGF to smooth muscle cells was demonstrated. Receptor concentration in calf‐aorta smooth muscle cells was higher in dense cultures compared to sparse cultures. The time course of binding and dissociation of 125I‐labelled EGF was similar in “dense” and “sparse” cultures.

Human uterine smooth muscle cells in culture exhibited a finite lifespan. There was no stimulation of DNA synthesis in response to serum and EGF in cells of high population doubling level (PDL); although 125I‐labeled EGF binding was higher in old cells (high PDL) compared to young cells (low PDL). This increase in binding was shown to be due to changes in the concentration of receptors without changes in their affinity for EGF.
Estrogen receptors and effects of estrogen on membrane electrical properties of coronary vascular smooth muscle
Authors: Harder David R. , Coulson Patricia B.
Published: February 2005

The effect of estrogen stimulation in vitro on the electrical properties of vascular smooth muscle (VSM), and the concentration of estrogen receptors in VSM were measured in isolated coronary arteries. Microelectrode measurements of the dog coronary artery membrane potential (Em) showed quiescent values of −51 millivolts (mV) and an input resistance (rin) of 10 megohms. Addition of diethylstibestrol (DES) at 10−6 M hyperpolarized the membrane to −64 mV and reduced in resistance (rin) to 5 megohms within 15 minutes. Extrapolation of the Em vs. log [K]o curve to zero potential gave similar values of [K]i of around 170 mM in both normal and DES treated muscles suggesting that the DES induced hyperpolarization is not due to increased Na‐K pump activity. The 0.5% ethanol vehicle alone had no effect on the membrane potentials. Tetraethylammonium ion (TEA) induced action potentials in the previously quiescent tissue. When DES was applied in the presence of TEA, the membrane potential increased and the action potential were abolished. Scatchard analysis of the estrogen receptor binding demonstrated both a high and a low affinity receptor for estrogen in the VSM. These data indicate that DES hyperpolarizes the VSM cells by a mechanism other than an increased Na‐K pump activity. The mechanism of this increased Em may be due to factors which increase K+ conductance either mediated directly through estrogen interaction with its cytosolic receptors or through some unidentified second mechanism.
Inhibition by aphidicolin of cell cycle progression and DNA replication in sea urchin embryos
Authors: Ikegami Susumu , Amemiya Shonan , Oguro Mieko , Nagano Hiroshi , Mano Yoshitake
Published: February 2005

We have recently found that aphidicolin, a tetracyclic diterpene‐tetraol produced by several fungi, blocks DNA synthesis of sea urchin embryos by interfering with the activity of DNA polymerase α. These cells fail to proliferate in the presence of aphidicolin.

In continuation of these studies, we determined the drug‐sensitive stage in the first cell cycle of the sea urchin Clypeaster japonicus embryo.

In continuous exposure to aphidicolin (2 μg/ml) from five minutes after fertilization, mitotic division of the embryo was completely suppressed. Embryos were exposed to the drug at progressively later intervals and their capability for cytokinesis was examined. Evidence was thereby obtained that aphidicolin acts at the S‐period to inhibit DNA synthesis resulting in developmental arrest of the embryo.
Oxocarboxylic acids, pyridine nucleotide‐linked oxidoreductases and serum factors in regulation of cell proliferation
Authors: McKeehan Wallace L. , McKeehan Kerstin A.
Published: February 2005

When serum is made rate‐limiting for clonal multiplication of human diploid fibroblasts, the presence of a 2‐oxocarboxylic acid in the medium becomes essential. The requirement is independent of the 20 amino acids and glucose. Glyoxylic, pyruvic, 2‐oxoglutaric, and oxalacetic acids are most effective. The types of 2‐oxocarboxylic acids that support multiplication are oxidized substrates for several, pyridine nucleotide‐linked intracellular oxidoreductases. The requirement is not satisfied by carboxylic acids, oxidized substrates for oxidoreductases that are not linked to pyridine nucleotides, or by nonspecific electron acceptors. The quantitative requirement for 2‐oxocarboxylic acids in cell multiplication is markedly affected by the concentration of serum proteins in the medium. Therefore, 2‐oxocarboxylic acid metabolism may be related to the mechanism by which serum growth factors regulate cell multiplication.
Effect of cytosine arabinoside triphosphate on the incorporation of 3H‐thymidine in mitochondria isolated from HeLa cells
Authors: Mattoccia L. Pica , Sorrentino R.
Published: February 2005

HeLa cell mitochondria were allowed to incorporate 3H‐thymidine in a cell free system and the effect of ethidium bromide, cytosine arabinoside and cytosine arabinoside triphosphate on the labeling of mitochondrial DNA was studied. The labeled products, isolated by sedimentation velocity in CsCl‐ethidium bromide two‐step gradients, showed similar sedimentation profiles as in vivo labeled mtDNA. Cytosine arabinoside triphosphate and ethidium bromide strongly inhibited the labeling of mitochondrial DNA, whereas cytosine arabinoside appeared to be much less effective. Tritiated deoxycytidine was found to be incorporated by isolated mitochondria, whereas cytosine arabinoside was shown to enter the mitochondrial acid‐soluble pool but not to be incorporated in acid‐insoluble form. These results are in agreement with the previously reported findings of in vivo experiments.
Effects of depletion of K+, Na+, or Ca2+ on DNA synthesis and cell cation content in chick embryo fibroblasts
Authors: Moscatelli David , Sanui Hisashi , Rubin A. Harry
Published: February 2005

Decreasing the K+ concentration of the medium from 5 mM to 0.59 mM decreased the K+ content of chick embryo fibroblasts to 22% of control values and increased the Na+ content to 820% of control values. The alteration of monovalent cation content occurred within two hours but had no effect on the rate of DNA synthesis, as measured by 3H‐thymidine incorporation, for at least 16 hours. By decreasing the Na+ concentration in the medium, a 50% reduction in cellular Na+ could be obtained with no effect on thymidine incorporation. Since these changes in cellular Na+ and K+ are much larger than any known to occur under physiological conditions but have no effect on thymidine incorporation, we conclude that Na+ and K+ do not play a critical role in determining multiplication rate.

Addition of 1.8 mM EGTA to cells in media containing 1.7 mM Ca2+ and 0.8 mM Mg2+ inhibited thymidine incorporation and sharply decreased cellular K+ and increased cellular Na+ content. However, there was no reduction in total cellular Ca2+ levels. Likewise, decreasing the Ca2+ concentration of the medium below 0.01 mM inhibited thymidine incorporation, decreased cellular K+ and Mg2+, and increased cellular Na+ but did not affect total cellular Ca2+ levels. Inhibition of DNA synthesis, therefore, could not be correlated with changes in cellular Ca2+ levels.
Replicative activity of isolated chromatin from proliferating and quiescent early passage and aging cultured mouse cells
Authors: Weisman‐Shomer P. , Kaftory A. , Fry M.
Published: February 2005

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparations from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5‐fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase β is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin‐directed replication is differently arrested in nondividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.
Specific cellular defects in patients with Fanconi anemia
Authors: Weksberg Rosanna , Buchwald Manuel , Sargent Patricia , Thompson Margaret W. , Siminovitch Louis
Published: February 2005

Measurements of plating efficiency, accumulation of metaphases and generation times have shown that fibroblast from patients with Fanconi anemia (FA) have decreased probability of completing a further division after successful mitosis. Thus FA cells show decreased growth rates and increased generation times. We have also measured the survival of FA fibroblasts and lymphoblasts after treatment with a variety of mutagens. All FA cells show an increased sensitivity to drugs such as MMC and psoralen plus long wave length UV which cause DNA interstrand crosslinks. FA strains show varying degrees of sensitivity to these drugs and the extent of this sensitivity seems to be characteristic of each patient. FA cells are equal to controls in their sensitivity to other alkylating agents such as ethyl methane sulfonate, N‐methyl‐N1‐nitro‐N‐nitrosoguanidine and actinomycin D. Both the decreased growth and increased drug sensitivity may result from defect in DNA replication or repair.
Defect in polyamine metabolism in a BHK cell mutant temperature‐sensitive for rRNA maturation
Authors: Levin Eugene G. , Clark Jeffrey L.
Published: February 2005

A mutant of BHK cells (ts422E) temperature‐sensitive for processing 32S rRNA to 28S rRNA (Toniolo et al., '73) also loses the ability to synthesize polyamines and 5.8S rRNA when shifted to the non‐permissive temperature (39°). The activity of several enzymes not involved with polyamine synthesis, methylation of 32S rRNA, and small nuclear RNA production are apparently unaffected after at least 24 hours at 39°. When cultures are returned to the permissive temperature (33°), polyamine synthesizing capacity returns to normal as mature rRNA production resumes.
A variant vascular endothelial cell line with altered growth characteristics
Authors: McAuslan B. R. , Reilly W.
Published: February 2005

A variant endothelial cell type was found to arise spontaneously from cultures of bovine aortal endothelial cells. This variant showed no contact inhibition and overgrew confluent cultures of wild‐type endothelial cells. Unlike other reported variants of this cell type produced by chemical mutagenesis or by withdrawal of polypeptide growth factor, this variant retained the capacity to synthesise factor VIII antigen, but showed no alteration from wild‐type in capacity to adsorb platelets. The variant also had an increased capacity to bind FITC‐conjugated con A to its surface.
Induction of an oligodendroglial enzyme in C‐6 glioma cells maintained at high density or in serum‐free medium
Authors: Maltese William A. , Volpe Joseph J.
Published: February 2005

The relationship between cell density and the activity of 2′:3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP), an enzyme believed to be specific to oligodendroglial cells and myelin in the brain, has been studied in cultured C‐6 glioma cells. Over a 12‐day period, the specific activity of CNP underwent a 4‐fold increase in conjunction with an increase in the cell density (total protein/flask) and a decline in the growth rate of the cultures. In contrast, the specific activity of Na+, K+‐ATPase was not influenced by cell density. Experiments with cultures seeded at different initial densities indicated that the increase in CNP activity coincided with the attainment of a specific cell density rather than with the length of time that the cells were maintained in culture. Arrest of cell proliferation in non‐confluent C‐6 cells by means of thymidine blockade was not sufficient to cause an increase in the activity of CNP; however, removal of serum from the culture medium resulted in a 3‐fold induction of the enzyme in the absence of a high degree of cell contact. The induction of CNP in cells maintained in serum‐free medium paralleled the development of a series of distinct morphological changes reminiscent of glial differentiation, which occurred within 48 hours after removal of the serum. Inhibition of protein synthesis by cycloheximide prevented the induction of CNP in serum‐free cultures. The demonstration that an enhancement of an oligodendroglial characteristic in C‐6 glioma cells can be obtained by growing the cells to high density or by removing serum from the medium, provides further support for the suggestion that these cells may be analogous to the glial stem cells present in the developing brain.
A synergistic effect of glucocorticoids and insulin on the differentiation of myoblasts
Authors: Ball Eric H. , Sanwal B. D.
Published: February 2005

Glucocorticoids and insulin were found to act synergistically to promote differentiation in some clones of the L6 rat myoblast cell line. Other hormone effects on these cells were investigated to determine the extent of the synergism. The insulin stimulation of sugar transport was unaffected by glucocorticoids although they did by themselves slightly enhance transport. Glucocorticoids were found to increase the adhesiveness of the cells–an effect not influenced by insulin. Cyclic AMP levels were found to peak just prior to the time of the onset of fusion and insulin broadened this peak, while the combination of both hormones further lengthened the time for which cyclic AMP levels remained elevated.
Studies of morphologically atypical (“sprouting”) cultures of bovine aortic endothelial cells. Growth characteristics and connective tissue protein synthesis
Authors: Cotta‐Pereira Gerson , Sage Helene , Bornstein Paul , Ross Russell , Schwartz Stephen
Published: February 2005

Morphologic and biochemical studies were performed on cultures of bovine aortic endothelial cells which had developed a second growth pattern that has been referred to as “sprouting” (Gospodarowicz and Mecher, '78; Schwartz, '78). These morphologically atypical cells undergrew the intact endothelial cell monolayer and appeared only after the cells had reached confluence. They were ultra‐structurally very similar to endothelial cells, but synthesized reduced amounts of fibronectin and a predominance of type I procollagen, rather than the types III and IV procollagens synthesized by monolayer endothelial cells.

It is suggested that these cells represent phenotypically altered endothelial cells that differ in biosynthesis of secreted proteins and display a reduced contact‐inhibition.
Induction of both rat and mouse lactate dehydrogenase in hybrids between inducible rat glioma and uninducible mouse neuroblastoma cells
Authors: Meyer Robert D. , McMorris F. Arthur
Published: February 2005

The specific activity of lactate dehydrogenase (LDH; EC 1.1.1.27) is induced two‐fold by l‐norepinephrine (NE) in C6TK− rat glioma cells, but not in NA mouse neuroblastoma cells or various other nonglial cells. Previous reports have shown that the induction is mediated by cyclic AMP (cAMP) and possibly protein phosphorylation, and that it requires RNA and protein synthesis. To study the block to LDH induction in nonglial cells, we hybridized C6TK− cells with NA cells and isolated a hybrid clone in which LDH is inducible by NE. Mouse and rat LDH from hybrid cells were separated by electrophoresis and quantitated by two independent methods, and it was found that mouse and rat LDH were induced equally when cells were exposed to NE. The results suggest that inducibility of LDH is not determined by a cis‐acting control at the gene level, but rather by the presence or absence of an earlier component in the cAMP‐mediated induction system, and that the induction system acts indiscriminately on all active LDH gene copies in the cell.
Multinucleation in mouse fibroblasts cultured in methocel medium
Authors: Ishii Kazuhiro
Published: February 2005

3T3‐4E cells formed multinucleate cells with high frequency when incubated in methocel medium. The experiment with hydroxyurea and the cytological observation of mitoses showed that multinucleate cells were produced by nuclear division in the absence of cytokinesis. When transferred onto a solid substratum, most of the multinucleate cells divided within seven hours into mononucleate cells through the process of cytoplasmic division, indicating that cell spreading induced cytokinesis. Other mouse fibroblast lines examined so far showed only the low frequency of multinucleation. These findings indicate that in 3T3‐4E cells cultivated in methocel, nuclear division occurred independently of cytokinesis, and that cytokinesis was also anchorage‐dependent. This system will be available for studying cytoplasmic division of mammalian cells.
Cell cycle‐related variations in UV damage and repair capacity in chinese hamster (CHO‐K1) cells
Authors: Collins A. R. S. , Downes C. S. , Johnson R. T.
Published: February 2005

UV damage to CHO cell DNA, measured by formation of thymine‐containing dimers, increases from mitosis to early S phase. Computer simulation of UV absorption by the DNA of an idealized CHO cell at different stages in the cell cycle resembles the cycle dependence of UV damage.

Incision at UV damage sites, measured by the accumulation of breaks in preexisting DNA during 30 minutes' post‐irradiation incubation with the DNA synthesis inhibitors 1‐β‐D arabinofuranosylcytosine and hydroxyurea, increases from mitosis to interphase. Analysis of the dose dependence of DNA break accumulation indicates that both the affinity of the endonuclease for dimer sites and the maximum enzyme activity at saturating levels of dimers are significantly lower in mitosis than in interphase.

The killing of CHO cells by UV is enhanced if repair is temporarily inhibited by ara C. The DNA gyrase inhibitor novobiocin prevents UV‐induced incision.
Differential expression of lectin receptors during hemopoietic differntiation: Enrichment for granulocyte‐macrophage progenitor cells
Authors: Nicola Nicos A. , Burgess Antony W. , Staber Fritz G. , Johnson Gregory R. , Metcalf Donald , Battye Francis L.
Published: February 2005

Molecular changes occur at the surface of hemopoietic cells during differentiation from progenitor cells to mature granulocytes and macrophages. The differential expression of surface carbohydrate residues has been probed using lectins and the results used to purify normal mouse granulocyte‐macrophage progenitor cells. Ten different lectins were screened for selective interaction with mouse hemopoietic colony‐forming cells (CFCs), using agglutination or a quantitative analysis of the number of fluoresceinated lectin molecules bound per cell using a fluorescence activated cell sorter (FACS). Pokeweed mitogen (PWM), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), and peanut agglutinin (PNA) preferentially bound to CFCs so that it was possible to enrich 4 to 10‐fold for these progenitor cells by sorting for the highly fluorescent cells. Further analysis of the low and high angle light scattering characteristics of the CFCs indicated that these cells were polydisperse, but could be enriched ten‐fold by selecting for cells with high intensity low angle (90°) scatter and low intensity high angle (90°) scatter. PWM gave the best enrichment (10 to 15‐fold) for adult bone marrow CFCs, for CFCs from fetal sources (fetal liver, fetal blood), and for CFCs from the spleens of mice injected previously with outer membrane lipoprotein from E. coli. Three parameter sorting for CFC using the FACS (low angle scatter, high angle scatter, and PWM‐fluorescence) resulted in large enrichment factors (16 to 50‐fold) for CFCs from all the above sources. Over 7% of the cells sorted from bone marrow, 10% of the cells sorted from post‐lipoprotein spleen, and 28% of the cells sorted from fetal peripheral blood were hemopoietic CFCs. Ninety percent of the cells in these fractions had the morphology of blast cells or myelocytes. Thus, it was possible to identify the morphological characteristics of the hemopoietic progenitor cells. Screening of other developmental systems using quantitation of fluorescence with lectins should prove of general value for the purification of selected differentiation states.
The apparent discrepancy of ouabain inhibition of cation transport and of lymphocyte proliferation is explained by time‐dependency of ouabain binding
Authors: Segel George B. , Lichtman Marshall A.
Published: February 2005

Mitogenesis of human blood lymphocytes in culture is inhibited by concentrations of ouabain that are approximately one order of magnitude lower than those that block Na and K transport. For example, the 50% inhibition (ID50) of Na‐K transport, 280 nM, is seven‐fold greater than the ID50 for RNA synthesis, DNA synthesis, or blastogenesis, ˜40 nM. Yet, inhibition of transport and consequent reduction in cell K is considered responsible for the effects of ouabain on mitogenesis. Since synthetic processes are assessed at least 24 hours after lymphocyte stimulation, this discrepancy could be explained by either 1) a progressive increase in K leak, or 2) a progressive inhibition of Na‐K transport by ouabain during 24 hours of PHA treatment. We found that the lymphocyte membrane leak rate of K increased immediately after PHA treatment but did not increase further from 4 to 24 hours. In contrast, the ouabain sensitivity of 42K uptake was markedly increased with time: ID50 for 42K uptake of 35 nM at 24 hours as compared to 280 nM at 30 minutes. Measurement of ouabain binding revealed a seven‐fold increase in the lymphocyte‐associated ouabain after 24 hours compared to binding at 1 hour. These data indicate that the dose response of ouabain inhibition of active K transport and lymphocyte proliferation are closely correlated if one considers the slow membrane binding of ouabain at low concentrations.
Murine sarcoma growth factors affect the growth and morphology of cultured mouse epithelial cells
Authors: Keski‐Oja Jorma , De Larco Joseph E. , Rapp Ulf R. , Todaro George J.
Published: February 2005

A mouse embryo epithelial cell line, MMC‐E, was used to study the effects of purified murine sarcoma growth factors (SGFs) on the growth of epithelial cells. Murine SGF was a partially purified preparation from the serum‐free culture media of mouse fibroblasts transformed by Moloney murine sarcoma virus. SGFs stimulated DNA‐synthesis in resting cells and induced them to grow to higher densities than control cells. With continued exposure to SGFs, MMC‐E cells lost their postconfluency inhibition of division and formed small expanding foci. When the SGFs were removed and the cells were subcultured, they regained their normal phenotype, showing that the effects of SGFs are reversible on these epithelial cells. SGFs could also stimulate the MMC‐E epithelial cells to grow in soft agar, like the syngeneic fibroblasts (MMC‐F) from the same mouse embryo, but slower than the control fibroblastic clone. Microgram quantitites of SGFs were needed to stimulate soft ager growth of MMC‐E cells. The results indicate that SGF can bring about a phenotypic change in the growth pattern of epithelial cells as well as fibroblastic cells.
The effects of 5‐bromodeoxyuridine on the growth and morphology of transformed rat liver cells
Authors: Avdalovic Nebojsa , Weibel Josef , Diamond Leila
Published: February 2005

The effects of bromodeoxyuridine (BrdUrd) on the growth, morphology, and tumorigenicity of the spontaneously transformed rat liver cell line R72/3 were studied. These cells grow either in suspension or in a monolayer and are tumorigenic. In monolayer cultures, cells treated with low concentrations (2.5 μg/ml) of BrdUrd were larger, more spread out, and more firmly attached to the substratum than were untreated controls. Treated cells failed to grow in suspension or on confluent monolayers of 3T3 cells and did not form colonies in soft agar. Scanning electron microscopy revealed extensive flattening of treated cells and a dramatic reduction in the number of microvilli on the cell surface. Transmission electron microscopy showed an increase in polyribosomes and rough endoplasmic reticulum, as well as an enlargement of endoplasmic reticulum cisternae and a complete absence of the bundles of intermediate size filaments that were conspicuous in untreated cells. The persistence of these changes required the continuous presence of BrdUrd in the medium. The effects of BrdUrd were readily reversed by withdrawal of BrdUrd and were not expressed in the presence of excess thymidine.
Lack of correlation between effects of tumor promoter TPA on plasminogen activator production, phosphatidyl choline synthesis, and hexose transport in mammalian cell culture systems
Authors: Plagemann Peter G. W. , Estensen Richard D.
Published: February 2005

We have investigated the effects of the tumor promoter 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) on plasminogen activator production, hexose transport and metabolism, and the incorporation of choline into the acid soluble pool and into phosphatidylcholine in suspension cultures of mouse L, mouse P388 leukemia, human HeLa, and Chinese hamster ovary cells, and in monolayer cultures of baby hamster kidney (BHK), mouse 3T3, mouse 3T6, and mouse P388D1 macrophage‐like cells. BHK, 3T3, P388D1, and P388 cells produced plasminogen activator constitutively, but no significant production was observed in the other cell lines. Plasminogen activator production was induced or stimulated by TPA only in P388 cells (10‐ to 20‐fold by 100 ng TPA/ml). On the other hand, phosphatidylcholine synthesis was stimulated by TPA only in HeLa cells, and hexose transport, as measured with 3‐0‐methyl‐D‐glucose, only in 3T3 and P388D1 cells, as well as in human lymphocytes. The stimulation of hexose transport occurred more rapidly than the induction of plasminogen activator production and seemed to be part of the mitogenic response of cells to TPA treatment. A stimulation of deoxyglucose uptake was similarly limited to 3T3 and P388D1 cells. A significant decarboxylation of carbon 1 of deoxyglucose occurred in P388 and P388D1 cells, but not in Novikoff cells, and any decarboxylation that occurred was not stimulated by TPA. The results indicate that the various investigated responses of cells to TPA are unrelated and occur independent of each other. The time courses of the biochemical responses also differ significantly.
Detection of lymphoid leukemia colony‐forming cells in abelson virus infected mice: Differences in inbred strains
Authors: Earl Christopher D. , Scher Charles D.
Published: February 2005

BALB/c or DBA/2 mice were infected with Abelson murine leukemia virus (A‐MuLV), pseudotype Molony murine leukemia virus (M‐MuLV). Infection of these mice with 104 focus‐forming units of A‐MuLV (M‐MuLV) induced overt leukemia, detectable grossly or microscopically in 90% of the mice at 20–38 days. However, these methods did not detect leukemia at 17 days or before. Bone marrow cells from A‐MuLV‐infected leukemic or preleukemic mice were placed in tissue culture in a soft agarose gel. Cells from leukemic or preleukemic BALB/c mice grew to form colonies of 103 cells or more, composed of lymphoblasts, whereas marrow cells from normal uninfected mice did not. Cells from these colonies grew to form ascitic tumors after intraperitoneal inoculation into pristane‐primed BALB/c recipient. Colony‐forming leukemia cells could be detected in the marrow of A‐MuLV‐infected mice as early as 8 days after virus incoluation. The number of colony‐forming leukemia cells increased as a function of time after virus inoculation.

Colony‐forming leukemia cells require other cells in order to replicate in tissue culture. Normal bone marrow cells, untreated or after treatment with mitomycin‐C, provide this “helper” function. Only in the presence of untreated or mitomycin‐C treated helper cells was the number of colonies approximately proportional to the number of leukemia cells plated.

Marrow cells from leukemic BALB/c mice form more colonies than those from leukemic DBA/2 mice. The number of colonies formed per 103 microscopically identifiable leukemia cells plated was determined to be 2–3 for leukemic BALB/c mice and 0.3 for DBA/2 mice. Cocultivation of leukemic DBA/2 marrow cells with mitomycin‐C treated normal BALB/c cells did not increase the number of colonies formed by the DBA/2 leukemic cells. Thus, the decreased ability of DBA/2 leukemia cells to form colonies appears to be a property of the leukemia cell population.
Augmentation of erythroid burst formation by the addition of thymocytes and other myelo‐lymphoid cells
Authors: Kanamaru Akihisa , Durban Elisa , Gallagher Michael T. , Miller Sandra C. , Trentin John J.
Published: February 2005

Bone marrow from barrier‐sustained specific pathogen‐free (SPF) CBA and C57BL/6 mice gave relatively low numbers of BFU‐E colonies in methylcellulose culture, as compared to conventional mice. Addition of thymocytes to the marrow cultures increased the yield of BFU‐E colonies more than fourfold in SPF mice but only 1.5‐fold in conventional mice. Colony size was also increased.

Increased yield of BFU‐E colonies was also obtained by co‐culture of bone marrow with lymph node cells or with bone marrow or spleen cells from 900R whole‐body‐irradiated mice. The effect appeared to be cellular rather than humoral. It was not reproduced by conditioned medium from thymus or pokeweed mitogen stimulated spleen cells. The helper effect of thymus cells was eliminated or reduced by freezing and thawing, or by 48 hours of incubation after irradiation. Treatment of bone marrow cells in vitro with anti‐theta serum and complement did not decrease the number of BFU‐E colonies. The putative helper cells appear not to be T cells, were non‐adherent to the plastic culture dish, and were cortisone resistant and radioresistant. The low BFU‐E colony yield from SPF mouse marrow is presumed to be largely the result of deficiency of these non‐T helper cells in SPF bone marrow, rather than of BFU‐E progenitor cells.
Insertion of muntjac gene segment into hamster cells by cell fusion
Authors: Tsukamoto Kyozo , Klein Richard , Hatanaka Masakazu
Published: February 2005

Indian muntjac diploid cells that have only three pairs of easily discernible large chromosomes were fused with hamster cells deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) using polyethylene glycol. Cells that survived in hypoxanthine‐aminopterine‐thymidine (HAT)‐oubaine medium were analyzed.

Hybrid cells containing both muntjac and hamster chromosomes in a given cell were not found. Instead, the cells had the same chromosomal sets as those of either parental muntjac or hamster cells.

A clonal isolate that had the same chromosomal sets as those of parental hamster cells was analyzed in detail and showed the following characteristics: (1) portions of the survival curve in various concentrations of HAT medium were intermediate between those of parental cells; (2) expressions of both muntjac and hamster antigen(s) were detected by immunofluorescence staining; (3) the mobility of the enzyme HGPRT in gel electrophoresis differed from that of parental hamster or muntjac cells. These results indicate that the clonal isolate (AD202h) is a somatic cell hybrid of hamster and muntjac that contains chromosomal sets of hamster with an inserted segment of the muntjac genome, including HGPRT.

The formation of such an unusal hybrid and a possible explanation of transfer of some gene segments in the hybrid cell in this system are discussed.
Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma‐derived cell lines
Authors: Axelrod Helena R. , Levine Arnold J.
Published: February 2005

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40‐transformed teratocarcinoma‐derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density‐inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.
Selective high metabolic lability of uridine triphosphate in response to glucosamine feeding of untransformed and polyoma virus‐transformed hamster fibroblasts
Authors: Rapaport Eliezer , Christopher C. William , Ullrey Donna , Kalckar Herman M.
Published: February 2005

Derepression of hexose transport in a line of Syrian hamster fibroblasts (Nil) and polyoma‐transformed (PyNil) hamster fibroblasts is obtained when cells are either starved for glucose or fed with fructose as the only hexose source. D‐glucosamine feeding of these cells does not alter the repressed state with regard to hexose transport. High, derepressed rates of galactose transport were changed to low, repressed rates, within 18 hours of refeeding glucose‐starved cells with D‐glucosamine as the only hexose source. Nil and PyNil cells, when cultured in the presence of D‐glucosamine, undergo rapid reductions in total cellular uridine 5′‐triphosphate (UTP) pool sizes. By contrast, the total cellular pools of adenosine 5′‐triphosphate, guanosine 5′‐triphosphate, and cytosine 5′‐triphosphate (ATP, GTP, and CTP) were only moderately affected by the treatment of the cells with glucosamine. The metabolic drain of the UTP pools in PyNil cells was much more pronounced than in the untransformed cells. The larger and more rapid metabolic lability of UTP pools in the transformed cells may be the primary reason for the selective toxicity of glucosamine on tumor cells. A comparison of the effects of glucosamine on hexose‐starved Nil and PyNil cells demonstrated that only the untransformed cells were able to utilize glucosamine to increase the hexose starvation‐depleted pools of all nucleoside triphosphates. Accumulation of UDP‐glucosamine and UDP‐N‐acetylglucosamine followed the reduction in the UTP pools. Inhibition of protein synthesis by cycloheximide during glucosamine feeding led to higher levels of UDP‐glucosamine and UDP‐N‐acetylglucosamine accumulation. It is suggested that the drain of UTP pools during glucosamine treatment proceeds through the formation of the UDP‐aminosugars which turn over due to the action of intracellular UDP‐aminosugar pyrophosphatase activities.
Cation transport in different volume populations of genetically low K+ lamb red cells
Authors: Lauf P. K. , Valet G.
Published: February 2005

During the first three months after birth lambs produce sequentially three erthryocyte populations of different mean volume as demonstrated by electric sizing methods (Valet, Franz, and Lauf, J. Cell. Physiol. 94 (1978) 215). We separated by centrifugal elutriation the small volume population (type II) red cells of a genotypically low K+ (LK) lamb from the population containing the larger volume type I and III cells, an admixture of fetal (I) and adult (III) erythrocytes. The cells were separated at various time intervals after birth and analyzed with respect to their volumes, cation contents, and cation flux properties by means of 86Rb uptake. The effect of anti‐L on K+ pump and leak fluxes was ascertained in unseparated and separated red cells. It was found that the small red cells of population II, transiently present for several weeks, were fully developed LK cells with K+ pumps responding characteristically to the stimulatory action of anti‐L. In constrast, the larger cells of population I and III were of high K+ (HK) nature at early time points, the K+ pump activities approximately ten times higher than adult LK cells. These cells constitute an admixture of type I fetal HK cells, and type III reticulocytes which are precursors for the final type III adult LK cells, since anti‐L had a small stimulatory effect. At later times, however, only adult type III LK cells predominated. The data directly support our earlier finding that the HK‐LK transition in genotypically LK lambs is primarily governed by cellular replacement.
Lymphocyte capping induced by polycationized ferritin
Authors: Butman Bryan T. , Bourguignon Gerard J. , Bourguignon Lilly Y. W.
Published: February 2005

In order to better understand the mechanism of lymphocyte surface receptor redistribution induced by externally added ligands, polycationized ferritin (PCF), a nonconventional ligand, was tested using both fluorescence and electron microscopy for its ability to cause patching and capping of anionic molecules on the surface of both transformed and normal mouse lymphocytes. Binding of PCF at 0°C for 1 hour induces the appearance of patches; subsequent incubation at 37° for 30–60 minutes causes the formation of a cap structure with the lymphoid cells tested (T‐lymphoma cells and splenic lymphocytes). Using various experimental treatments (e.g., sodium azide, cytochalasin B and D, colchicine, prefixation, and cold temperatures), PCF‐induced capping has been found to be temperature sensitive, and to require metabolic energy and an intact cytoskeletal system. In addition, using double immunofluorescence techniques which involve rhodamine‐labeled PCF and fluorescein‐conjugated heavy meromyosin, it has been observed that the formation of the PCF‐induced cap coincides with an accumulation of intracellular actin directly beneath the cap structure. Furthermore, agents such as dibutyryl cyclic AMP and theophylline, which cause an increase in intracellular cyclic AMP, have been shown to stimulate PCF‐associated capping. This study suggests that increasing levels of intracellular cyclic AMP may activate, directly or indirectly, membrane‐associated contractile elements required for the aggregation of membrane proteins into patches and caps.
Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells
Authors: Borghetti Angelo F. , Piedimonte Giuseppe , Tramacere Mariarosaria , Severini Alberto , Ghiringhelli Paolo , Guidotti Guido G.
Published: February 2005

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly+ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transportspecific model substrates. L‐Proline, 2‐aminoisobutyric acid, and glycine were primarily taken up by system A; L‐alanine and L‐serine by system ASC; L‐phenylalanine by system L; and L‐lysine by system Ly+ in SV3T3 cells. L‐Proline and L‐serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na+‐dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na+‐independent systems L and Ly+remained substantially unchanged. The density‐dependent change in activity of system A occurred through a mechanism affecting transport maximum (Vmax) rather than substrate concentration for half‐maximal velocity (Km). Transport activity of systems A and ASC was severalfold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus‐transformed 3T3 cells that have lost density‐dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.