bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA synthesis and proliferation of human lymphocytes in vitro: III. Fate of cycling cells in aging cultures of phytohemagglutinin stimulated human lymphocytes
Authors: Mendelsohn John , Hudig Dorothy , Castagnola Janice
Published: February 2005

There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA‐stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse‐labeled with 3H‐thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G2 + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non‐viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA‐stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G2 phases. These observations provide evidence against the existence of a specific “restriction point” in G1 or at the G1/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G2 phases retain the capacity to complete the cell cycle in more favorable culture environments.
Passive membrane permeability to small molecules and ions in transformed mammalian cells: Probable role of surface phosphorylation
Authors: Makan Nizar R.
Published: February 2005

Addition of ATP to medium surrounding intact, transformed 3T3 cells causes the formation of aqueous channels in the plasma membrane. This effect of extracellular ATP is sharply dependent on the pH and temperature of the incubation medium, and is inhibited by low levels of La3+ or ruthernium red; inhibition is also obtained with concentrations of Mg2+ ions that exceed a ratio of Mg/ATP of one. The effect of ATP on membrane channel formation is unaffected by chelators of metal ions or by prior modification of the cell surface with various surface‐active enzymes or sulfhydryl reagents. Under conditions which favor aqueous channel formation, incubation of intact 3T6 cells with ATP (γ‐32P) leads to phosphorylation of two membrane components with apparent molecular weight of 40,000 (40K) and 110,000 (110K) daltons; the 110K component which is unaffected by trypsin under normal conditions is rendered trypsin‐sensitive by the phosphorylation reaction, probably as a result of a conformational change. Conditions which inhibit aqueous channel formation also inhibit phosphorylation of the 110K protein and decrease the labeling of the 40K component. These results indicate the probable role of cell surface phosphorylation, involving one or both of these components, in the formation of aqueous channels in transformed 3T3 cells. Aqueous channel formation by extracellular ATP is not associated with gross unfolding of the cell surface as revealed by lactoperoxidase‐catalyzed iodination of the 3T6 cell surface.
Transmembrane potential changes during phagocytosis in rat alveolar macrophages
Authors: Miles P. R. , Bowman L. , Castranova V.
Published: February 2005

Studies were carried out to measure changes in the transmembrane potential of rat alveolar macrophages during exposure of the cells to zymosan particles or to the membrane perturbant, phorbol‐12‐myristate‐13‐acetate (PMA), and to determine if changes in membrane potential are related to superoxide anion release. Exposure of the cells to either zymosan or PMA leads to membrane depolarization, which precedes superoxide anion release. Furthermore, the magnitude of the depolarization is dependent upon the concentration of either zymosan or PMA. During exposure of the alveolar macrophages to increasing levels of zymosan, there is an increase in the amount of superoxide released as well as an increase in the magnitude of the depolarization. Incubation of the cells in medium containing 150 mMK+, a medium which causes membrane depolarization, leads to superoxide release from resting cells and a decrease in the amount of superoxide released from cells exposed to zymosan. These results indicate that release of superoxide anion from rat alveolar macrophages is related to membrane depolarization and suggest that the transmembrane potential change may act as a signal to initiate the phagocytotic responses of the cells.
Studies of the copper‐binding proteins in Menkes and normal cultured skin fibroblast lysates
Authors: LaBadie Gundula U. , Beratis Nicholas G. , Price Peter M. , Hirschhorn Kurt
Published: February 2005

Proteins of approximately 10,000 daltons (presumably metallothionein) and greater than 75,000 daltons bound 64Cu when this metal was added to fibroblast lysates. Treatment with either 2‐mercaptoethanol or the disodium salt of ethylenediamine tetraacetic acid demonstrated that the high molecular weight copper‐binding proteins in lysates prepared from both normal and Menkes fibroblasts exhibited a relatively low affinity for copper compared to the 10,000 dalton protein(s). No difference was detected in the affinity of the low molecular weight protein(s) of normal and Menkes fibroblast lysates for copper. The amount of 64Cu bound to the 10,000 dalton protein(s), however, was approximately two to three times greater in lysates prepared from Menkes fibroblasts than from normal fibroblasts. Mixing experiments indicated that the increased binding of 64Cu to the 10,000 dalton protein(s) in lysates of Menkes fibroblasts did not result from the deficiency of a factor that effects the cleavage of copper from this protein(s), from the presence of a soluble inhibitor, or from the lack of an activator. In addition, the use of lysates, rather than whole cells, demonstrated that the observed differences in copper binding between the normal and the Menkes fibroblasts were not caused by an abnormality in the membrane transport of copper in the mutant cells. Thus the findings suggest that the increased accumulation and the reduced efflux of copper previously observed in cultured Menkes fibroblasts result either from an increased amount of the 10,000 dalton copper‐binding protein(s) or from an increased capacity of this molecule(s) for copper.
Amiloride‐resistant Madin‐Darby Canine kidney (MDCK) cells exhibit decreased cation transport
Authors: Taub Mary , Saier Milton H.
Published: February 2005

Cation transport systems were investigated in mutant Madin‐Darby Canine Kidney (MDCK) cells resistant to the diuretic drug amiloride. The mutants were isolated previously as clones resistant to the cytotoxic effects of 3 × 10−4 M amiloride. Decreased amiloride transport by the Na+ channel was implicated as the basis of the resistance (Taub, '78). Consistent with this hypothesis, Na+ accumulation was lower in amiloride resistant cells than in normal sensitive MDCK cells. Kinetic studies indicated that Na+ uptake in MDCK cells occurs by a single ATP independent transport system—the Na+ channel.

In several amiloride‐resistant clones, including clone Amr2, the decreased Na+ uptake was associated with a decrease in both the Km and Vmaxfor Na+ uptake by the Na+ channel. In Amr2 cells no significant alteration in the inhibitory effect of amiloride on Na+ uptake was observed. As the Na+ channel may actually be a general uptake system for monovalent cations (a number of cations inhibit Na+ uptake), the uptake of these inhibitory cations was examined in Amr2 cells. Both Ca++ and ouabain‐insensitive Rb+ uptake occurred at decreased rates in Amr2 cells as compared with normal MDCK cells. However, further uptake studies suggested that Na+, Ca++ and ouabain‐insensitive Rb+ uptake all occur by different systems. Thus several transport systems may be defective in Amr2 cells. Amr2 cells were also resistant to the inhibitory effects of amiloride on CO2 evolution from pyruvate. These observations indicate that alterations at a number of molecular sites may be associated with defective Na+ transport via the Na+ channel in amiloride‐resistant cells. Thus the amiloride‐resistant cells are potentially valuable in examining the interrelationships between Na+ transport and other cellular functions.
Sodium and rubidium uptake in cells transformed by Rous sarcoma virus
Authors: Bader John P. , Okazaki Takumi , Brown Nancy R.
Published: February 2005

Rates of uptake and intracellular concentrations of monovalent cations were measured in virus‐transformed and nontransformed chick embryo (CE) cells. Uptake of 22Na+ into cells transformed by the BH strain of Rous sarcoma virus (RSV‐BH) (CE‐BH) was about double the rate of uptake into CE cells, or cells transformed by the Schmidt‐Ruppin strain (RSV‐SR): CE‐SR. Likewise, the rate of efflux of 22Na+ was greater in CE‐BH cells than in CE or CE‐SR cells. The greater permeability of CE‐BH cells to Na+ was apparent in higher intracellular Na+ concentrations. Experiments with cells exhibiting temperature‐dependent transformation showed that new RNA and protein synthesis was a requirement for the acquisition of increased Na+ permeability, suggesting that the change is an indirect effect of the virus‐coded transformation‐inducing protein. Rates of 86Rb+ uptake, used as a measure of K+ influx, were indistinguishable in CE, CE‐BH, and CE‐SR cells. Also, equilibrium intracellular levels of 86Rb+ were similar in transformed and nontransformed cells, as were observed concentrations of K+. Also, no differences in ATPase activity, as indicated by ouabain binding or temperature sensitivity, were observed. We conclude that monovalent cations play no direct role in RSV‐induced transformation, although the higher levels of Na+ in CE‐BH cells may be responsible for other distinguishing biochemical features of these cells.
Sulfhydyl group involvement in the modulation of neutral amino acid transport in thymocyte membrane vesicles
Authors: Kwock Lester
Published: February 2005

Membrane vesicles from rat thymocytes accumulate 2‐aminoisobutyric acid in the presence of 0.1 M NaCl. Uptake is half maximal between 15 and 30 seconds after addition of the amino acid and reaches a plateau value after about 2 minutes. The uptake of 2‐aminoisobutyric acid can be modulated by various sulfhydryl reagents. Reduced glutathione leads to an inhibition of uptake whereas oxidized glutathione increases uptake. Agents such as insulin and diamide which can induce disulfide formation lead to an activation of transport. These data indicate that uptake of the Na+‐dependent amino acid, 2‐aminoisobutyric acid, in thymocytes is modulated by a putative plasma membrane, sulfhydryl‐containing protein.
Regulation of cell division and of tyrosinase in B16 melanoma cells by imidazole: A possible role for the concept of metabolite Gene regulation in mammalian cells
Authors: Montefiori David C. , Kline Ellis L.
Published: February 2005

Results of hemacytometer cell counts and of tyrosinase measurements made by the Pomerantz method demonstrate that imidazole added to the medium of cultured B 16 mouse melanoma cells can stimulate tyrosinase specific activity and inhibit cell division. These effects are greater than with adenosine 3′,5′ cyclic monophosphate (cAMP) or the cAMP‐phosphodiesterase inhibitor theophylline. The effects of imidazole on cell division and tyrosinase are enhanced by theophylline and antagonized by cAMP. Cyclic AMP‐phosphodiesterase activity in cell‐free extracts can be inhibited by theophyllne and stimulated by imidazole. However, imidazole does not affect cAMP‐phosphodiesterase specific activity in vivo, nor does it affect intracellular cAMP concentrations as determined by competitive protein‐binding assays. In contrast, the specific activity of cAMP‐phosphodiesterase in vivo is stimulated by cAMP and theophylline, supporting the hypothesis that cAMP and agents which increase intracellular cAMP concentrations induce the synthesis of cAMP‐phosphodiesterase. Studies with actinomycin‐D and cycloheximide support the hypothesis that cAMP can also mediate posttranslational activation of tyrosinase. Similar experiments suggest that imidazole, or a derivative therof, can induce the synthesis of tyrosinase at the pretranslational level of control. We hypothesize that this type of regulation (pretranslational) by imidazole may define a role for the concept of “Metabolite Gene Regulation” (MGR), in mammalian cells.
Fluorimetric measurements and chromatin condensation patterns of nuclei from 3T3 cells throughout G1
Authors: Moser Gertrude C. , Fallon Robert J. , Meiss Harriet K.
Published: February 2005

Using two cytological methods based on nuclear morphology, quinacrine dihydrochloride (QDH) staining and premature chromosome condensation (PCC), it has been possible to identify cell cyle positions within G1 of growing and arrested 3T3 cells. The fluorescent intensity of QDH‐stained interphase cells appears to decrease as the cells pass from mitosis to S phase. Likewise, the length and thickness of prematurely condensed chromatids can be related to the cells' position within the G1 period. Data are presented that deal with three interrelated topics: (1) We determined by fluorometric measurements of nuclei from 3T3 cells that the visual observation of the decrease in QDH fluorescence during G1 reflects an actual decrease in total fluorescence and not a dispersion of the fluorescent chromatin in a larger nuclear area. (2) We correlated the results obtained by QDH staining with those of PCC on the same cell samples blocked in G1 by different conditions. Serum‐starved and contact‐inhibited cell nuclei had the highest intensity, hydroxyurea‐treated ones had the lowest intensity, while that of isoleucine‐deprived cells was in between. The same relative order of G1 positions was obtained based on PCC morphology. Thus, both methods monitor the state of chromatin condensation and can be used to identify cell cycle position within G1.(3) We showed with both methods that the states of chromatin resulting from the various G1 blocking conditions differ from each other.
Synthesis of a metallothionein‐like protein in cultured human skin fibroblasts: Relation to abnormal copper distribution in Menkes' disease
Authors: Bonewitz Roland F. , Howell R. Rodney
Published: February 2005

A metallothionein‐like protein (MTP) is synthesized in normal diploid human skin fibroblasts cultured in Zn‐ or Cu‐supplemented medium. Synthesis of MTP is not detected in cells cultured without metal supplementation of complete tissue‐culture medium. Cultured fibroblasts from patients with Menkes' disease accumulate excess Cu which chromatographs both with highmolecular‐weight protein(s) and with a Cu‐MTP. Under normal culture conditions, the Menkes' MTP incorporates [35S]‐cystine, but not appreciable amounts of 65Zn. However, Menkes fibroblasts retain the ability to incorporate 65Zn into MTP in response to Zn supplementation of the medium. The results do not support the idea that Menkes' disease results from a failure of Cu to bind to MTP, but rather that an elevated intracellular Cu concentration in Menkes' disease fibroblasts leads to association of excess Cu with high‐molecular‐weight protein, stimulating synthesis of a Cu‐binding MTP.
Modulation of functional and optimal (Na+‐K+)ATPase activity during the cell cycle of neuroblastoma cells
Authors: Mummery Christine L. , Boonstra Johannes , Van Der Saag Paul T. , De Laat Siegfried W.
Published: February 2005

Functional and optimal activities of the (Na+‐K+)ATPase, as determined by ouabain‐sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro‐2A) cells. The cells were synchronized by selective detachment of mitotic cells.

The ouabain‐sensitive K+ influx decreased more than fourfold from 1.62 ± 0.11 nmoles/min/106 cells to 0.36 ± 0.25 nmoles/min/106 cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 ± 0.30 nmoles/min/106 cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 ± 0.25 nmole/min/106 cells in early S phase to 2.10 ± 0.92 nmoles/min/106 cells just prior to the next mitosis. The ouabain‐insensitive component rose linearly through the cycle in the same manner as the protein content/cell.

Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid‐S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis.

The (Na+‐K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed.

Complete inhibition of the (Na+‐K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+‐K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.
The reversible inability of isoproterenol‐activated parotid gland cells to initiate DNA synthesis in the hypocalcemic thyroparathyroidectomized rat
Authors: Tsang B. K. , Whitfield J. F. , Rixon R. H.
Published: February 2005

An intraperitoneal injection of the beta‐adrenergic drug dl‐isoproterenol hydrochloride (100 mg/kg body weight) into male (190–210 g) albino rats caused two cyclic AMP surges (peaking at 10 minutes and again between 8 and 12 hours) and the initiation of DNA synthesis (between 16 and 20 hours) in the parotid glands. The parotid cells in hypocalcemic thyroparathyroidectomized rats still responded to isoproterenol injection by generating the two cyclic AMP surges, but they did not initiate DNA synthesis unless a blood calcium‐elevating combination of parathyroid hormone (50 USP units/100 g of body weight) and 1α,25 (OH)2 vitamin D3(200 pmoles/100 g of body weight) was injected along with the isoproterenol.
Unbalanced growth and cell death in HeLa S3 cultures treated with DNA synthesis inhibitors
Authors: Frankfurt Oskar S.
Published: February 2005

Flow cytometry indicated that significant amounts of dsRNA were accumulated in HeLa S3 cells blocked at or near G1/S boundary by hydroxyurea (HU) or excess thymidine (TdR). The dsRNA/DNA ratio increased in these cells in a manner characteristic of unbalanced cell growth. In HU‐treated cells, dsRNA content was maximal 16 hours after addition of the drug and did not change significantly during the next 24 hours. The DNA content in blocked cells increased by 10%. Cell viability assessed by colony formation in soft agar decreased exponentially in HU‐treated cultures after 16 hours of incubation. Correlation between loss of cell viability and rate of cell proliferation after removal of HU was observed, as determined by cell count and analysis of cell cycle progression. In TdR‐treated cultures cells slowly progressed into mid S‐phase during 40 hours and dsRNA accumulation continued during this period. Cell viability was not significantly affected by treatment with excess TdR, indicating that unbalanced growth per se, as measured by dsRNA accumulation, is not lethal for the cells. After reversal of DNA synthesis inhibition by removal of the drug, cells treated with HU for 16 hours or TdR for 16–24 hours promptly progressed through the cell cycle. This progression was accompanied by accumulation of significant amounts of dsRNA. As a result, cells in G2 phase had a very high dsRNA content leading to retention of the unbalanced condition (increased dsRNA/DNA ratio) in the daughter cells. It is suggested that dsRNA accumulation in the cell is controlled to a certain degree by cell progression through the S phase. This type of control, evidently, was reflected in limited dsRNA accumulation in the cells blocked at or near G1/S border, in continuous dsRNA accumulation in the cells slowly progressing through S phase, and in accumulation of large amounts of dsRNA after renewal of progression through the S phase.
Isolation and preliminary characterization of bleomycin‐resistant mutants from Chinese hamster ovary cells
Authors: Akiyama Shin‐Ichi , Kuwano Michihiko
Published: February 2005

Stable mutants resistant to an anticancer antibiotic, bleomycin‐A2, were selected in Chinese hamster ovary (CHO) cell either spontaneously or after ethylmethane sulfonate mutagenesis. Fluctuation analysis showed that bleomycin resistance occurs in CHO at a rate of 6.50–6.58 × 10−7 mutations per cell per generation. Bleomycin‐A2‐resistant cell lines exhibited increased resistance to bleomycin analogs—bleomycin‐A5, ‐B2, ‐B4, and pepleomycin. Colchicine, mitomycin C, and ultraviolet light irradiation inhibited colony formation equally in CHO cells and in bleomycin‐resistant mutants. Cell‐cell hybridization tests showed that bleomycin‐resistance behaves as a dominant trait. Bleomycin‐in‐activating activity in the mutant cell extracts was three to fourfold higher than that in extracts of the parental CHO cell.
Glucocorticoids inhibit the stimulatory effect of epidermal growth factor on the initiation of DNA synthesis
Authors: Otto Angela M. , Natoli Clara , Richmond K.M. Veronica , Iacobelli Stefano , De Asua Luis Jimenez
Published: February 2005

Confluent, quiescent Swiss 3T3 cells in culture can be stimulated to initiate DNA synthesis and divide by addition of growth factors to the culture medium. Here we show that hydrocortisone and other steroids which have glucocorticoid activity inhibit the stimulation of these cells by epidermal growth factor (EGF) in contrast to their reported enhancement of stimulation by fibroblast growth factor (FGF). Binding studies using [3H]‐triamcinolone acetonide show that Swiss 3T3 cells contain a single class of glucocortioid receptor of uniform affinity (KD = 2.0 nM), and about 34,000 receptor sites per cell. Those steroids which displace bound [3H]‐triamcinolone acetonide are also effective in inhibiting the stimulation of DNA synthesis by EGF in the presence or absence of insulin, and the concentration of triamcinolone acetonide required for one‐half maximal biological effect is in the same range as the KD. A similar concentration is required for one‐half maximal enhancement of the effect of FGF. These results suggest that both the inhibitory and stimulatory effects of glucocorticoids may be mediated via these receptors, the different effects thus being due to differences in the intracellular events triggered by each growth factor.
The effect of calcium chelation on lymphocyte monovalent cation permeability, transport and concentration
Authors: Quastel Michael R. , Segel George B. , Lichtman Marshall A.
Published: February 2005

We have quantified the effect of EGTA on K exodus and uptake in human blood lymphocytes. When lymphocytes were exposed to a medium containing an EGTA concentration that resulted in an ionized Calcium (Ca) of less than 10 μM, K exodus began to increase. This increase reached nearly threefold that of the control rate in a medium containing sufficient EGTA to reduce the ionized Ca concentration below 0.1 μM. When K exodus was increased, K uptake increased proportionately. This increase in K uptake represented active transport and was associated with an 80% increase in intracellular Na concentration from 15 to 27 mM. The addition of Ca to a medium containing EGTA reversed to normal the increased K exodus and uptake. Histidine, a potent chelator of divalent cations other than Ca, had no effect on K transport. These data indicate that extracellular Ca chelation leads to an increase in lymphocyte membrane permeability and cation leak. This increased leak is associated with an elevation of the cell Na and an increase in transport to a rate equivalent to that of the exodus rate. The compensatory increase in active transport maintains the cell monovalent cation concentration within 10 to 15 mM of unperturbed levels.
Comparison of the control and pathways for degradation of the acetylcholine receptor and average protein in cultured muscle cells
Authors: Libby Peter , Goldberg Alfred L.
Published: February 2005

In the studies reported here, we investigated whether the degradation of the acetylcholine receptor (AChR) in cultured muscle cells involves similar mechanisms as and is controlled in a manner similar to, the catabolism of the bulk of cell protein. We compared these processes after labeling cell protein with radioactive leucine or phenylalanine for 24 hours, or labeling the acetylcholine receptor with (125I)‐bungarotoxin. The apparent average half‐life of cell protein was 38 ± 2 hours and that of the receptor‐toxin complex was 25 ± 1 hours. Incubation in media lacking serum and embryo extract accelerated the degradation of both average protein and the receptor‐toxin complex. Insulin reduced the rate of catabolism of both average protein and the receptor‐toxin complex toward levels seen in the presence of serum. However, although these two degradative processes seem to be controlled similarly, they probably involve different mechanisms. The protease inhibitors leupeptin and chymostatin, which slowed overall proteolysis in nongrowing muscles and hepatocytes, reduced the degradation of the ACh receptor by 2–11‐fold, but had no, or only slight, effects on the catabolism of average protein, even when overall proteolysis was accelerated by omitting serum and embryo extract. Chloroquine, an inhibitor of lysosomal function, also reduced the degradation of AChR, by about 10‐fold, but decreased overall protein breakdown by only 20–30%. Incubation of myotubes at lower temperatures reduced both degradative processes, but affected the breakdown of the receptor to a greater extent. Thus the rate‐limiting steps in these processes have different activation energies. Incubation with 2‐deoxyglucose, an inhibitor of glycolysis, decreased the breakdown of average protein but not that of the receptor‐toxin complex. However, the two degradative processes were sensitive to azide, an inhibitor of oxidative phosphorylation. Although the lysosome is the primary site for AChR degradation and perhaps for degradation of other surface proteins, the breakdown of most proteins in myotubes seems to involve a distinct proteolytic system requiring metabolic energy.
The expression of glial fibrillary acidic protein in a rat cerebellar cell line
Authors: Giotta Gregory J. , Cohn Melvin
Published: February 2005

A rat cerebellar cell line, WC5, derived by transformation with Rous sarcoma virus, which is temperature‐sensitive for transformation (ts‐RSV), can be induced to express glial fibrillary acidic protein (GFAP). Immunofluorescence, radioimmune assay, and electron microscopy studies show that GFAP is expressed in WC5 cells grown at the nonpermissive temperature (NPT), but not at the permissive temperature (PT) for transformation. GFAP is first detectable about 3 days after incubating cells at the NPT, and reaches an apparent plateau by the seventh or eighth day. The expression of GFAP is reversible; shifting cells from the NPT to the PT causes a dramatic decrease in GFAP after 96 hr. In order to determine if the expression of GFAP is linked to the temperature‐sensitive transforming activity of the viral src gene product, phenotypic revertants of WC5 were established. By the criteria of morphology and growth in agar, the revertant lines, in contrast to the parent cell line WC5, were shown to exhibit a transformed phenotype at both the NPT and PT. Immunofluorescence studies on several of the revertant cell lines show that they do not express GFAP at either the PT or NPT. These findings suggest that the expression of GFAP in WC5 is linked to the expression of the src gene product. The advantage of using ts‐RSV to derive neural cell lines which exhibit differentiated properties is discussed.
Nutrient transport in a bovine lens epithelial cell line
Authors: Prasad Rajendra , Shopsis Charles , Southern A. Louis , Hochstadt Joy
Published: February 2005

A bovine calf lens epithelial cell line (CLE‐1) that synthesizes crystallin has been established in culture and some of its transport properties have been characterized using both cells and membrane vesicles derived from them. The membrane vesicles fractionate with high recovery of plasma membrane markers, showing a 40‐fold purification of 5′‐AMPase and a 20‐fold decrease in the specific activity of the mitochondrial marker enzyme succinic dehydrogenase relative to a cell homogenate. Transport sites demonstrated higher specific activity than has been seen in vesicles from cell lines studied previously. The uptake of α‐amino isobutyric acid (AIB) (an alanine analog) by CLE‐1 cells is stimulated four‐ to fivefold by Na+ and exhibits a Km of 5.4 mM with a Vmax of 50 pmoles/min μg of cell protein. The uptake of leucine was not Na+ stimulatable. The uptake of AIB by the cells was reduced by 43% at confluence. Thus, the cell density dependent behavior of the uptake of the alanine amino acid family in CLE‐1 is similar to that of various fibroblast cells.

The Na+ caused a threefold stimulation of AIB uptake in the membrane vesicles, while vesicular uptake of leucine was unaffected by Na+. The uptake of adenine, guanine, uridine, and guanosine was also tested in these vesicles. The substrates were rapidly accumulated, came to a steady state distribution within 1–2 minutes, and were recovered as the unaltered compounds after uptake.
The role of energy in hyperthermia‐induced mammalian cell inactivation: A study of the effects of glucose starvation and an uncoupler of oxidative phosphorylation
Authors: Haveman J. , Hahn G. M.
Published: February 2005

When cultured Chinese hamster cells were exposed to 43°C hyperthermia, effects due to glucose deprivation and to the presence of the uncoupler of oxidative phosphorylation, carbonylcyanide‐3‐chlorophenylhydrazone, during the 43°C treatment proved to be strongly accelerated compared to the effects at normal temperature (37°C). This strongly indicates that the availability of energy plays an important role in the response of these cells to hyperthermia. One of the reasons cells die after hyperthermia may be a lethal lack of energy. Cells heated before glucose deprivation were able to maintain viability for a longer period during deprivation than cells without the preheat treatment. As the cells might develop thermotolerance after the heat exposure, this suggests that cells in the thermotolerant state use energy in a more economical way.
Properties of a growth factor activity present in crude extracts of rat uterus
Authors: Sirbasku David A. , Leland Frances E. , Benson Robert H.
Published: February 2005

We have shown previously (D.A. Sirbasku, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:3786–3790) that an estrogen‐inducible growth factor activity for rat mammary and rat pituitary tumor cells can be identified in extracts of rat uteri, although at the time of that report only a limited biochemical characterization of the activity was presented. In this report, we have evaluated the growth factor activity for lipid, steroid hormone or protein‐like properties. Uterine growth factor activity was assayed by measure of the increased cell number of the MTW9/PL rat mammary tumor cell line established by this laboratory and described previously (D.A. Sirbasku, 1978, Cancer Res. 38:1154–1165). Studies showed the following characteristics of growth factor activity: destroyed by trypsin treatment; labile when heated at 80°C; partially denatured by 6 M guanidine or 8 M urea treatment or 50% aqueous solutions of organic solvents; inactivated by extremes of pH or overnight treatment with mild acid; not dialyzable at neutral pH; of apparent molecular weight of 70,000 daltons by G‐100 Sephadex chromatography; possessing an isoelectric point of 4.8 to 5.2; not chloroform/methanol extractable; and not in any way identified as either a lipid or a steroid hormone. The data available suggest that the uterine growth factor activity is a protein or polypeptide of apparent high molecular weight, and that this activity does not directly correspond to other known growth factors.
Reverse transformation of vole cells transformed by avian sarcoma virus containing the src gene
Authors: Puck Theodore T. , Erikson Raymond L. , Meek William D. , Nielson Stanley E.
Published: February 2005

Vole cells transformed by avian sarcoma virus carrying the src gene lose their fibroblastic morphology, the organized cytoskeletal system of the normal fibroblastic cell, the typical fibronectin deposit around the cell membrane, and the ability to shut off multiplication when suspended in liquid medium. All of these transformation characteristics are reversed by treatment with cAMP derivatives. Moreover, the cAMP treatment does not cause loss of activity of the src gene product. These data imply that cAMP exerts its effect at or after the point in the metabolic pathway affected by the src gene product, pp60src. Presumably, the decision to adopt the transformed or the normal state is determined by the degree to which the src gene or cAMP‐mediated kinase activities respectively predominate in the cell. The development of all four transformation characteristics as a result of introduction of the src gene, and their coordinate reversal by cAMP derivatives, supports the previous thesis that in the normal vole or CHO fibroblast all four properties are part of a common regulatory system.
Commitment and proliferation kinetics of human lymphocytes stimulated in vitro: Effects of α‐MM addition and suboptimal dose on concanavalin a response
Authors: Hall David J. , O'Leary James J. , Sand Theodore T. , Rosenberg Andreas
Published: February 2005

We have examined the effect of alpha‐methylmannoside (α‐MM) addition to concanavalin A (con A)‐stimulated peripheral human lymphocytes. With a previously established kinetic model, we have, from the time course of proliferation, extracted the responding clone size, rate of entry of this clone into S phase, and the length of the lag period. We have studied the effect of con A dose and time of addition of α‐MM to optimally stimulated cells on these kinetic parameters. We show that neither a low dose of con A nor an early addition of ‐‐MM to optimally stimulated cells results in a change in the responding clone size. That is, all of the potentially responsive cells appear to become “committed” to enter the cell cycle regardless of the presence of α‐MM early in the culture or in the presence of suboptimal stimulation. However, the rate at which these committed cells enter the first S phase is a function of the dose of con A and time of addition of α‐MM and varies over a wide range. It is the variation in this parameter that accounts for virtually all of the diminished response previously interpreted as a time‐dependent irreversible commitment of mitogen‐stimulated cells. The previous work using only fixed time points for measuring thymidine uptake and the concept of commitment must be reevaluated in light of the kinetic evidence presented.
Hexose sugar transport activity in a mouse fibroblast cell line temperature sensitive for expression of the transformed phenotype
Authors: Karrs Thomas M. , Quinlan Dennis C.
Published: February 2005

A temperature‐sensitive mouse fibroblast cell line was used to examine the relationship between hexose sugar uptake rates and the control of cell growth. The cell line used (ts‐H6‐15) is a derivative of SV‐3T3 cells, exhibiting a transformed phenotype at 32°C and a normal phenotype at 39°C. For cells actively growing at either temperature, a marked decrease in the rate of 3‐0‐methyl‐D‐glucose (3‐0‐MeG) transport is observed as cell population density increases. At all cell population densities tested, 3‐0‐MeG transport rates (at a common assay temperature) were greater in H6‐15 cells grown at 32°C than at 39°C, with the enhancement being maximal at the lowest cell densities. The effect of low serum‐arrest on H6‐15 cells revealed that cells growing at 39°C arrest in G1, while cells at 32°C stop more randomly throughout their cycle. Under conditions of low serum‐arrest the rate of 3‐0‐MeG transport remained as high as in actively growing cells at both 32°C and 39°C. However, 2‐deoxyglucose uptake rates were growth state‐dependent at 39°C, indicating perhaps metabolic as well as membrane‐level control of sugar accumulation. These results further demonstrate that rates of hexose sugar transport by themselves are not always absolutely correlated with rates of cell proliferation and, thus, may not be reliable predictors of cell growth potential.
Sensitivity of excision repair in normal human, xeroderma pigmentosum variant and Cockayne's syndrome fibroblasts to inhibition by cytosine arabinoside
Authors: Cleaver James E.
Published: February 2005

Inhibition of the gap‐filling, polymerizing step of excision repair by 1‐β‐D‐arabinofuranosylcytosine (ara‐C) after irradiation with ultraviolet light in human diploid fibroblasts resulted in the formation of persistent DNA strand breaks in G1, G2, and plateau phase cells, but not in S phase cells. Addition of hydroxyurea to ara‐C resulted in partial inhibition of repair in S phase cells. These observations can be explained either in terms of changing roles in repair for different DNA polymerases throughout the cell cycle or by the presence of a pool of deoxycytidine nucleotides during S phase equivalent to an external source of deoxycytidine at 50 μM concentration. A similar concentration dependence on ara‐C was observed for inhibition of repair in normal human, xeroderma pigmentosum (XP) variant, and Cockayne's syndrome cells. Ara‐C produced a similar number of breaks in normal and Cockayne's syndrome cells but slightly more in XP variant cells. Exonuclease III and S1 nuclease independently both degraded about 50% of the 3H‐thymidine incorporated into repaired regions in the presence of ara‐C. Sequential digestion with both enzymes degraded nearly 90% of the repaired regions. These observations can be explained if excision repair proceeds by displacing the damaged strand so that both the 3H‐labeled patch and the damaged region are still ligated to high molecular weight DNA and compete for the same complementary strand during in vitro incubation with the nucleases. The amount of 3H‐thymidine incorporated in DNA by repair decreased with increasing concentrations of ara‐C and hydroxyurea, suggesting that the incomplete patches became shorter under these conditions. Extrapolation of the digestion kinetics with exonuclease III permits an estimate of the normal patch size of about 100 nucleotides, consistent with previous estimates.
Clonal variation in cultured neuroblastoma cells. I. Isolation and characterization of variants
Authors: Lombardo J. M. , Lanks K. W.
Published: February 2005

Two spontaneously arising variant clones were selected from the N18 neuroblastoma cell line solely on the basis of their flattened morphology and tight adherence to the culture flask. Two other clones having the round loosely adherent morphology typical of the parent line were also selected, and flat variants were shown to arise in them upon prolonged cultivation. The flat variant clones have slower growth rates in culture, lower cloning efficiencies in suspension, and reduced acetylcholinesterase inducibility when compared with either the parent N18 line or the round cell clones. Cells of both morphologic types have high levels of plasminogen activator and are tumorigenic, although the variants have a slower growth rate in vivo, consistent with their slower growth rate in culture. SDS‐polyacrylamide gel electrophoresis of total protein from the two cell types shows that the flat variants have increased amounts of a 200,000 molecular weight polypeptide that has tentatively been identified as the heavy chain of myosin. Round morphological revertants from one of the flat variant clones exhibited growth characteristics typical of the parent N18 line, but their content of myosin heavy chain, although reduced, was not so low as that in the round cell clones originally isolated. The possibility of a causal relationship between flat morphology, reduced suspension cloning efficiency, and increased content of myosin heavy chain is discussed.
The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells
Authors: Tobler Jerry , Krieger Monty , Stroud Robert M.
Published: February 2005

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells was studied using 125I‐labeled canine plasminogen. Throughout a 3‐day period, 125I‐plasminogen in the incubation medium bound to the cells and was degraded, first to intermediate‐sized macromolecules that were the same size as the large (74,600‐dalton) and small (25,000‐dalton) chains of active plasmin, and to smaller fragments including 3‐iodo‐L‐tyrosine. Binding to SV3T3 cells was independent of the protease‐dependent morphological change (PDMC)1 characteristic of these and many other transformed cells. The SV3T3, and to a somewhat lesser extent, the 3T3 cells, both accumulated and released into the incubation medium 3‐iodo‐L‐tyrosine, a terminal lysozymal digestion product.

The results of a sublethal cell‐surface trypsinization assay suggest that the cell‐associated plasminogen was primarily bound to the surfaces of the 3T3 and SV3T3 cells while the macromolecular degradation products including active plasmin were inside the cells. The rate of 125I plasminogen degradation exhibited by SV3T3 cells was approximately two times greater than that of 3T3 cells, which presumably reflects differences in endocytosis or lysosomal hydrolysis, or both. The rates were unaffected by addition of pancreatic or soybean trypsin inhibitor sufficient to inhibit PDMC.

In the incubation medium, plasminogen was activated to plasmin by SV3T3, but not by 3T3 cells. However, 95–100% of plasmin covalently bound to a 47,000‐dalton canine serum component, which could be dissociated from plasmin by hydroxylamine: 95–100% of the plasmin was inactive to reaction with DF32P. Thus the serum component is a plasmin inhibitor. The plasmin‐containing complex in the medium had an apparent molecular weight of 212,000. Under denaturing conditions, the complex dissociated into two covalently modified plasmin‐containing species of 153,000 and 127,000 daltons. In addition to forming a complex with a serum component, the plasmin is cleaved into two small fragments (∼10,000 and 12,000 daltons) by as‐yet uncharacterized serum factors.
The effect of added H1 histone and polylysine on DNA synthesis and cell division of cultured mammalian cells
Authors: Kundahl E. , Richman R. , Flickinger R. A.
Published: February 2005

Addition of H1 histone or polylysine (10 μg/ml) to cultured Friend erythroleukemia cells or to two mouse lymphoma cell lines (el‐4 and S‐49) increased levels of cell division in these cultures. There is a stimulation of incorporation of labeled thymidine into DNA in cultures containing H1 histone and polylysine. DNA fiber autoradiographic experiments revealed that replicon size is decreased in the cells cultured with H1 histone and polylysine at later periods of culture.
The effect of reagents that increase membrane fluidity on the activity of 3‐hydroxyl‐3‐methyl glutaryl coenzyme a reductase in the CHO‐K1 cell
Authors: Sinensky Michael , Kleiner Jeffrey
Published: February 2005

The compounds cetyl trimethyl ammonium bromide (CTAB) and ethanol both decrease the order parameter of a spin probe embedded in cholesterol‐lecithin liposomes, but CTAB produces lowering of the order parameter comparable to that produced by ethanol at a 10,000‐fold lower concentration. Treatment of CHO‐K1 cells with CTAB or ethanol at concentrations that produce comparable increases of membrane fluidity produce a 2‐ to 3‐fold increase of microsomal membrane cholesterol to phospholipid ratio and a 2‐ to 3‐fold increase of the activity of the rate‐limiting enzyme of cholesterol biosynthesis, 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase. Cells treated with CTAB or ethanol show a progressively decreasing capacity to accumulate α‐aminoisobutyric acid with increasing drug treatment, but cells pre‐treated with CTAB are relatively resistant to the effects of CTAB on α‐aminoisobutyrate transport. The increase in HMG‐CoA reductase by CTAB or ethanol is not observed when these compounds are added directly to cell extracts but, rather, is only observed after 8 hours of exposure of intact cells to these drugs. Actinomycin D and cycloheximide treatment prevent the increase in enzyme activity, and the increase is also blocked in a regulatory mutant of the CHO‐K1 cell with permanently repressed HMG‐CoA reductase activity.

These data are consistent with a homeoviscous adaptation mechanism in the CHO‐K1 cell, in which increased activity of HMG‐CoA reductase, through a process requiring RNA and protein synthesis, compensates for conditions that increase membrane fluidity by increased cellular cholesterol biosynthesis and cholesterol to phospholipid ratio.
Differences in oxygen‐dependent regulation of enzymes between tumor and normal cell systems in culture
Authors: Simon Lawrence M. , Robin Eugene D. , Theodore James
Published: February 2005

Metabolic studies in tumor cells have indicated that bioenergetic regulatory mechanisms geared to acute changes in oxygen availability are abnormal. In the present studies we have examined bioenergetic adaptations to chronic oxygen depletion in culture maintained tumor cells in comparison to normal cell lines. Activities of two key glycolytic enzymes (pyruvate kinase (PyKi) and phosphofructokinase (PFK)) were measured in two tumor cell lines (fibrosarcoma (FS) and Hela) and two normal cell lines (rat lung fibroblasts (RLF) and WI‐38) maintained in culture for up to 96 hours under aerobic (PO2 ∼ 140) and hypoxic PO2 ∼ (15) conditions. Exposure to low O2 tensions for 96 hours resulted in significant increases in PyKi and PFK in both RLF and WI‐38, but did not alter activities of these enzymes in either FS or HeLa cell systems. Activities of two enzymes involved in O2 metabolism (cytochrome oxidase (CyOx)) and superoxide dismutase (SOD) were also measured in the two tumor cell lines and in RLF. Chronic hypoxia significantly decreased the activities of CyOx and SOD in RLF cell systems but did not alter the activities of these enzymes in the tumor cells. In these studies, the tumor‐derived cell lines do not demonstrate specific enzymatic responses to sustained oxygen depletion in vitro noted in normal cell systems, suggesting significant abnormalities in regulatory mechanisms geared to chronic changes in molecular O2.