bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of growth in vitro by glucocorticoids in mouse embryonic facial mesenchyme cells
Authors: Salomon David S. , Pratt Robert M.
Published: February 2005

The growth of primary embryonic facial mesenchyme cells established from cleft palate sensitive A/J and resistant C57BL/6J (C57) mice is inhibited by glucocorticoid treatment. A reduction in cell number in both A/J and C57 culture is accompanied by a significant decrease in [3H] thymidine incorporation into both acid soluble and insoluble material. No significant changes in total cellular protein or [14C] leucine incorporation were observed in either cell type. A greater reduction in [3H] thymidine incorporation occurs in cells undergoing exponential growth following steroid exposure than in cells approaching stationary growth. In both A/J and C57 cultures the reduction in cell number exhibits a dose‐dependent response to dexamethasone; is specific for glucocorticoids; and is dependent upon the concentration of serum in which the cells are maintained. A/J cells show a greater sensitivity to the inhibitory effect of dexamethasone on cell number and thymidine incorporation than comparably treated C57 cells. Specific, high affinity, saturable cytoplasmic receptors for [3H] dexamethasone are present in the maxillary cytosols from which the primary cultures were established. These receptors exhibit binding specificity for glucocorticoids, and have properties which are similar to glucocorticoid receptors identified in other systems. In both cell types, a correlation exists between the degree of growth inhibition or reduction of [3H] thymidine incorporation and the level of glucocorticoid receptors. These results provide evidence for a receptor‐mediated set of responses to glucocorticoids in these cells.
Physiological quiescence in plasma‐derived serum: Influence of platelet‐derived growth factor on cell growth in culture
Authors: Ross Russell , Nist Cynthia , Kariya Beverly , Rivest Mary Jane , Raines Elaine , Callis Jim
Published: February 2005

A platelet‐derived growth factor can be shown to be the principal stimulant of DNA synthesis in whole blood serum for those cells that require serum for maintenance and growth in culture. Cell free plasma‐derived serum lacks such platelet‐derived material. 3T3 cells and primate arterial smooth muscle cells can be maintained in a quiescent state in culture for as long as six weeks in plasma‐derived serum. Such cells can grow logarithmically after exposure to 5% whole blood serum or as little as 100 ng/ml of partially purified platelet factor. The cell cycle of smooth muscle cells has been studied in the quiescent (5% plasma‐derived serum) and growing state (5% whole blood serum or 5% plasma‐derived serum plus platelet factor). The generation time of smooth muscle cells is 16 to 18 hours as shown by autoradiographic frequency of labelled mitoses. The generation time is the same for cells in the growth fraction in either 5% whole blood serum or 5% plasma‐derived serum. Thus, platelet factor acts by recruiting cells into the growth fraction rather than effecting a change in the duration of the cell cycle. Flow microfluorimetry studies on cells growing logarithmically in 5% whole blood serum give the following phase durations:

G1 = 5.6 hours; S = 7.6 hours; and G2 + M = 3.8 hours.

Based on these studies the argument is presented that cells cultured in 5% plasma‐derived serum provide a more physiological base for the study of quiescence than do cells in low concentrations of whole blood serum or confluent, density inhibited cells at high (5% or greater) concentrations of whole blood serum. Furthermore, 5% plasma‐derived serum represents an appropriate state to examine the perturbation of quiescent cells.
Lymphocyte response to phytohemagglutinin: Intracellular volume and intracellular [K+]
Authors: Holian A. , Deutsch C. J. , Holian S. K. , Daniele R. P. , Wilson D. F.
Published: February 2005

The effect of phytohemagglutinin (PHA) on lymphocytes was examined with respect to free intracellular water volume and intracellular [K+]. At a cell concentration of 30 × 106 lymphocytes/ml in modified Hank's Buffered Salt Solution (HBSS) in the presence of 10% human AB serum, addition of PHA at 3 mg/ml resulted in a 24–27% decrease in free intracellular water space within 30 to 60 minutes and a return to control level after three hours. A larger change in intracellular water (44%) was observed under similar conditions in the absence of serum. The absolute intracellular K+ content did not change after PHA addition, but the cell water volume decrease arising from PHA addition resulted in a 29% increase in intracellular [K+] at 60 minutes. The decrease in lymphocyte water volume induced by PHA was also observed for concanavalin A which stimulates lymphocyte proliferation, but not for wheat germ lectin, an agglutinating agent which is not mitogenic. Thus, volume regulation may be closely associated with the mitogenicity of these compounds.
Superoxide dismutase specific activities in cultured human diploid cells of various donor ages
Authors: Duncan Michael R. , Dell'orco Robert T. , Kirk K. D.
Published: February 2005

It has been postulated that superoxide dismutase (SOD) protects cells from free radical‐induced damage. In these experiments SOD specific activity was measured as established human diploid cell lines from various donor ages progressed through their in vitro lifespans. Significant elevations in activity occurred during the in vitro lifespans of cells from fetal and newborn donors, but no change in activity was detected during the lifespan of cells from an adult donor. In addition, a direct relationship between enzyme activity and donor age was detected with the following relative activities: adult > newborn > fetal. The possible relationship between these findings and the free radical theory of aging is discussed.
Selective suppression of the transcription of ribosomal genes in mouse‐human hybrid cells
Authors: Perry R. P. , Kelley D. E. , Schibler U. , Huebner K. , Croce C. M.
Published: February 2005

Mouse‐human hybrid cells that preferentially segregate either mouse or human chromosomes were analyzed for their relative content of mouse and human rRNA genes and for their capacity to transcribe these genes. A distinctive Hind III restriction fragment containing 28S rRNA sequences was used to distinguish between mouse and human rDNA and a set of distinctive loop structures in the 45S pre‐rRNA was used to distinguish between mouse and human gene transcripts. Our results indicate that the genes of only one species are transcriptionally active in these hybrid cells, even though both sets of genes are present.
Intracellular proteolysis in rat cardiac and skeletal muscle cells in culture
Authors: Mayorek Nina , Pinson Arie , Mayer Michael
Published: February 2005

Treatment of adult rats with dexamethasone resulted in an increase in cardiac muscle weight but a decrease in skeletal muscle weight. The different response of skeletal and cardiac muscles to the glucocorticoid was also reflected by a dexamethasone‐induced enhancement of myofibrillar protease activity in the gastrocnemius muscle and an inhibition of a similar proteolytic activity in the heart. Newborn rats also exhibit the same, tissue‐specific response to the glucocorticoid hormone. Consequently, the difference between cardiac and skeletal muscle responsiveness to conditions of wasting was investigated in culture. Average rates of degradation of intracellular proteins were determined in cultured cells derived from rat skeletal and cardiac muscle by following the release of radioactivity from cells prelabelled with 14C‐phenylalanine. The release of label into the TCA soluble medium as measured during 12 hours of incubation, conformed to a first‐order reaction and both cell types were found to degrade intracellular proteins at a similar rate. After 12 hours of incubation in a complete Ham F‐10 medium supplemented with serum approximately 18% of total cellular protein was degraded. Incubation in a minimal medium or serum‐deprivation enhanced the average rate of proteolysis to a value of 29% degradation at 12 hours indicating that intracellular proteolysis in these cells is responding to nutritional deprivation by increased activity. However, addition of glucose (22.2 nM) or dexamethasone (10−6M) to the incubation medium failed to affect the rate of net protein degradation. Under no experimental condition could a difference be found between the proteolytic response of skeletal muscle cells to that of cardiac muscle cells and both cell types displayed similar changes in rates of protein degradation under various nutritional and hormonal conditions in culture. Thus, protein sparing in the heart of intact animals under catabolic conditions which enhance protein loss in skeletal muscle can probably not be ascribed to intrinsic differences in the direct response of cellular proteases to the tested hormones and nutrients. Rather, an extracellular factor(s) is apparently required for induction of the differential response of these tissues in the intact animal to protein wasting conditions. Alternatively, cells in culture might have lost the property of differential degradative response which operates in vivo.
Abnormal regulation of de novo purine synthesis and purine salvage in a cultured mouse T‐cell lymphoma mutant partially deficient in adenylosuccinate synthetase
Authors: Ullman B. , Clift S. M. , Cohen A. , Gudas L. J. , Levinson B. B. , Wormsted M. A. , Martin D. W.
Published: February 2005

The isolation and characterization of a mutant murine T‐cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU‐100, was isolated from a mutagenized populatio of S49 cells by virtue of its resistance to 0.1 mM 6‐azauridine in semisolid agarose. The AU‐100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine.

High performance liquid chromatography of AU‐100 cells extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3‐fold over those levels found in wild type cells. The AU‐100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which as in wild type cells is diminished by incubation of AU‐100 cells with adenosine. However AU‐100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells.

In other growth rate experiments, the AU‐100 cell line was shown to be resistant to 6‐thioguanine and 6‐mercaptopurine. Levels of hypoxanthine‐guanine phosphoribosyltransferase (HGPRTase) measured in AU‐100 cell extracts, however, are 50‐66% greater than those levels of HGPRTase found in wild type cell extracts. Nevertheless this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in vivo.

The AU‐100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine necleotides.
Stimulation of hexose transport in cultured human skin fibroblasts by insulin
Authors: Germinario Ralph J. , Oliveira Maureen
Published: February 2005

The effect of insulin on hexose transport in cultured human skin fibroblasts.

Studies were carried out on cultures of human skin fibroblasts to explore the effect of insulin on hexose transport in serum‐starved monolayers. Insulin (100 mU/ml) stimulated 2‐deoxy‐D‐glucose transport (30% above control values) after 30 minutes exposure time, the response being similar up to four hours exposure to insulin. In several experiments (n = 22) employing three cell strains, insulin (100 mU/ml) exposure led to variable stimulation of 2‐deoxy‐D‐glucose transport (an average of 37% above control values, with a range of 0 = 120%). The insulin‐induced stimulation of 2‐deoxy‐D‐glucose transport showed a dose dependency with increasing amounts of insulin, the response being maximal at an insulin concentration of 100 mU/ml. Kinetic analysis of 2‐deoxy‐D‐glucose transport showed that insulin addition resulted in a slight change in the transport Km (3.13 to 4.06 mM) and a 1.8‐fold increase in the transport Vmax (17.6 nanomoles/mg protein/min to 32.1 nanomoles/mg protein/min). Insulin also stimulated the transport of 3‐0‐methyl‐D‐glucose while the hexokinase activity of the cells was not affected. Further, this insulin‐induced stimulation of sugar transport was not blocked by cycloheximide. The results indicate that insulin stimulated the stereospecific carrier‐mediated of hexose transport in cultured human skin fibroblasts.
Stimulation of chondroitin sulfate synthesis by β‐D‐xyloside in chondrocytes of the proteoglycan deficient mutant nanomelia
Authors: Stearns K. , Goetinck P. F.
Published: February 2005

The potential of nanomelic chondrocytes to synthesize chondroitin sulfate was investigated by providing the mutant cells with p‐nitrophenyl‐β‐D‐xyloside, a compound which acts as an artificial acceptor for glycosaminoglycan synthesis. Under these conditions the synthesis of chondroitin sulfate in nanomelic and normal chondrocytes is comparable. The chondroitin sulfate synthesized by the mutant is indistinguishable in molecular size and composition from that synthesized by similarly treated normal chondrocytes.
Enhanced sterol synthesis in concanavalin A‐stimulated lymphocytes: Correlation with phospholipid synthesis and DNA synthesis
Authors: Chen S. Shi‐Hua
Published: February 2005

Incorporation of (14C)choline and (3H)myo‐inositol into the total lipid fraction, incorporation of (14C)acetate into the sterol fraction and incorporation of (3H)thymidine into DNA were studied in human lymphocyte cultures. Concanavalin A induced an increase in the incorporation of these labels with the following features: (a) Phospholipid synthesis was increased promptly. The lag time for the increase in sterol synthesis and DNA synthesis were 5 hours and 27 hours respectively; (b) The increase in phospholipid synthesis and sterol synthesis was proportional to ConA concentration initially. Cells treated with a high concentration of ConA showed very low levels of DNA synthesis; (c) The increase in phospholipid synthesis could be abolished immediately by α‐Methyl‐Mannoside. α‐Methyl‐Mannoside blunted but did not abolish the increase in sterol synthesis. α‐Methyl‐Mannoside enhanced DNA synthesis of those cells which had been treated by a high concentration of ConA; and (d) Selective inhibition of sterol synthesis with 25‐hydroxycholesterol did not prevent the increase in phospholipid synthesis, but it blocked the increase in DNA synthesis. Supplement of LDL, HDL or total lipoproteins to lymphocyte cultures was effective in preventing the inhibition of DNA synthesis by 25‐hydroxycholesterol. These results suggest that in lymphocyte activation by ConA phospholipid synthesis, sterol synthesis and DNA synthesis were sequentially increased. The rate of cellular commitment to mitogenesis was proportional to ConA concentrations. High concentrations of ConA arrested the cell growth at a postcommitment point in the G1 phase. Enhanced phospholipid synthesis was a precommitment event. Enhanced sterol synthesis was a postcommitment event and reflected the requirement of an increased cholesterol supply for the passage of cell growth through G1.
Measurement of total, intracellular and surface bound cations in animal cells grown in culture
Authors: Sanui Hisashi , Rubin A. Harry
Published: February 2005

A procedure is described in which animal cells grown in culture on a dish are rapidly rinsed in situ with 0.25 M sucrose solutions for subsequent measurement of total, intracellular and rapidly exchangeable Na+, K+, Mg2+ and Ca2+ by atomic absorption spectrophotometry. Repeated rinses with CO2‐free (pH ∼7) 0.25 M sucrose solution produced essentially no loss of cellular protein or cations. One 10‐second rinse with CO2‐saturated (pH 4) 0.25 M sucrose solution removed a rapidly proton exchangeable cellular cation fraction which is interpreted as being externally (membrane) bound. Rinses with physiological electrolyte solutions are shown to produce loss of cellular protein as well as displacement of surface exchangeable cations. Thus, isotonic sucrose solution is more satisfactory than electrolytic media for rinsing cultured cells prior to measurement of cellular cations. The technique employing sucrose rinse media is very rapid and reproducible and permits measurement of total, intracellular or surface bound Na+, K+, Mg2+ and Ca2+ in the same sample.
The chemotactic factors induced movement of calcium and sodium across rabbit neutrophil membranes: Effect of desensitization to cytochalasin B
Authors: Naccache P. H. , Showell H. J. , Becker E. L. , Sha'afi R. I.
Published: February 2005

The preincubation of rabbit neutrophils with the chemotactic factor F‐Met‐Leu‐Phe and the subsequent addition of cytochalasin B has previously been shown to induce a time, concentration and calcium dependent loss of secretory responsiveness in neutrophils. This has been termed desensitization. The results reported here first confirm that lysosomal enzyme release from neutrophils will still occur in the absence of extracellular calcium. In addition, a time dependent decrease in the magnitude of the cytochalasin B induced influxes of 45Ca and 22Na was found upon preincubation with F‐Met‐Leu‐Phe. In the presence of extracellular Ca2+, this decrease in ionic responsiveness reaches a maximum by five minutes preincubation with F‐Met‐Leu‐Phe. In the absence of added extracellular Ca2+ an initial and rapid (<1 minute) loss of ionic responsiveness is followed by partial recovery as the length of the preincubation with the chemotactic factor is increased from one to five minutes. These changes in ionic responses correspond exactly to the changes in secretory behavior of the neutrophils. Desensitization can thus be explained on the same ionic basis as that underlying the secretory response of the neutrophils. In addition, these results provide information about the sequence of events involved in the cytochalasin B and chemotactic factor induced release of lysosomal enzymes in neutrophils.
Electron probe microanalysis of elemental composition of mouse cardiac myocytes during post‐natal maturation
Authors: Dykes Sandra Goles , Cameron Ivan L. , Smith Nancy K. R.
Published: February 2005

Elemental concentrations in the cytoplasm and nucleus of post‐natal mouse myocytes were measured at 2, 4, 8, 16, 32 days and in adults using electron probe X‐ray microanalysis. Using an analysis of variance test, significant age dependent changes were found in intracellular potassium, sulfur, phosphorus, and chlorine concentations (mg/kg dry weight), while sodium and magnesium concentrations did not show significant changes. The application of t tests following linear regression analyses (age versus concentration for each element) did, however, give a significant slope for cytoplasmic sodium, potassium, sulfur, and phosphorus values. The findings correspond closely with an emission spectroscopy‐titrimetric study of whole heart ventricle of the same developmental period (Hazelwood and Nichols, '70).
The role of Heme in the regulation of the late program of friend cell erythroid differentiation
Authors: Mager Dixie , Bernstein Alan
Published: February 2005

The addition of a chemical inducer, such as dimethylsulfoxide (DMSO), to cultures of mouse Friend erythroleukemic cells results in the induction of a number of late erythroid events, including the accumulation of globin mRNA, the induction of hemoglobin synthesis, the appearance of erythrocyte membrane antigens (EMA), and the cessation of cell division. The experiments presented in this study demonstrate that heme is necessary but not sufficient for the loss of proliferative capacity associated with DMSO‐induced Friend cell differentiation, whereas the accumulation of globin mRNA and EMA can occur in the absence of heme synthesis or heme itself. These conclusions were reached by selectively inhibiting heme synthesis in DMSO‐treated cells in two independent ways: (i) Inducible cells were treated with 3‐amino‐1,2,4‐triazole (AT), a drug which inhibits the induction of heme synthesis in Friend cells in a dose‐dependent manner. Treatment of inducible Friend cells with 1.5% DMSO for five days caused the plating efficiency in methyl cellulose to decrease to 1% of that in untreated cultures. However, treatment of the cells with DMSO plus AT almost totally prevented this decrease in plating efficiency. The addition of exogenous hemin, which alone had no significant effect on plating efficiency, largely reversed the effect of AT in DMSO‐treated cells, reducing the plating efficiency to below 5%. In contrast to the marked effects of AT on the proliferative capacity of differentiating Friend cells, the levels of globin mRNA and EMA were only partially decreased in cells treated with DMSO plus AT, compared to cells treated with DMSO alone. (ii) The relationship between heme synthesis, terminal cell division, and the induction of globin mRNA was investigated further through the use of non‐inducible Friend cell variant clones. One such non‐inducible clone, M18, appears to be a phenotypic analog of inducible cells treated with DMSO plus AT. Clone M18 did not accumulate heme or hemoglobin, as detected by benzidine staining, nor lose its proliferative capacity in response to DMSO. However, globin mRNA was induced by DMSO in this clone. Treatment of clone M18 with DMSO plus hemin overcame the block in hemoglobin accumulation suggesting that M18 has a defect in the induction of heme biosynthesis. In addition, exposure of M18 cells to DMSO plus hemin caused a gradual decrease in plating efficiency which was not due to non‐specific toxicity. Prior incubation of M18 cells in DMSO for three to five days was necessary before hemin caused a rapid loss of proliferative capacity. Thus, these results, in agreement with the AT studies on inducible Friend cells and previous studies on the induction of EMA in clone M18, indicate that there may be both heme‐dependent and heme‐independent events in the program of Friend cell differentiation.
Application of the principles of enzyme kinetics to clonal growth rate assays: An approach for delineating interactions among growth promoting agents
Authors: Lechner John F. , Kaighn M. Edward
Published: February 2005

The interaction of mitogenic factors on a single cell type and the comparative activity of a given factor in diverse cell types have been studied byapplying the principles of Michaelis‐Menten kinetics to clonal growth data. Such comparisons are facilitated by derivation of two parameters; K, the mitogen concentration that gives half‐maximal clonal growth and a theoretical maximal growth rate, R. Both parameters are analogous to the Km and VMAX as applied to enzymatic reactions. Use of these parameters permits meaningful comparisons between cells with different growth rates.

Using kinetic analysis of dose‐response data, we found that normal human epithelial cells require 200 times more fetal bovine serum protein (FBSP) than a malignant line to multiply at their respective half‐maximal rates. Further, the K of normal cells was reduced to that of the malignant line by the inclusion of growth factors (EGF or FGF, and hydrocortisone) in the medium. On the other hand, even though greater levels of serum were required when growth factors and hydrocortisone were not present, their inclusion did not alter R. Interactions between mitogenic factors were shown to be unidirectional. Although EGF reduced the K, FBSP did not change the K. The same type of analysis revealed that hydrocortisone, which potentiated the mitogenic activity of EGF did not change the K. Kinetic analysis of cell growth should prove useful in studies on the relation between growth and tumor promotion as well as in the evaluation of growth‐inhibiting chemotherapeutic agents.
Rapid changes in poly (A)(+) mRNA content in growth stimulated fibroblasts following perturbations in protein synthesis
Authors: Meister Richard K. , Hulman Sonia E. , Johnson Lee F.
Published: February 2005

We have previously shown that when resting 3T6 cells are serum stimulated in the presence of inhibitors of protein synthesis, poly(A)(+) mRNA content increases extremely rapidly relative to cells stimulated in the absence of drug. Poly(A)(+) mRNA content nearly doubles within two hours, but then remains constant for at least ten hours (Johnson and Meister, '77). In this report we show that continuous exposure to both serum and cycloheximide are required to maintain this elevated mRNA level. Removal of either leads to an equally rapid decrease in poly(A)(+) mRNA content. If cycloheximide is withdrawn at either two or ten hours following serum stimulation in the presence of the drug, allowing the rapid (< 30 minutes) restoration of the rate of protein synthesis, we observe that poly(A)(+) mRNA content decreases within two hours to a level nearly equal to that found in resting cells prior to stimulation. If the drug is withdrawn but the serum stimulus is not, the rapid decrease in poly(A)(+) mRNA content is followed by an increase which is parallel to that which occurs in cultures stimulated in the absence of drug, but displaced from the latter by an interval approximately equal to the length of exposure of the drug. These results show that the mammalian cell is able to decrease as well as increase its content of poly(A)(+) mRNA in response to drug induced perturbations in the rate of protein synthesis. The changes in poly(A)(+) mRNA content occur extremely rapidly and may represent an attempt by the cell to correct the perturbation.
Spontaneous, mutagen‐induced and adenovirus‐induced anchorage independent tumorigenic variants of mouse cells
Authors: Bellett A. J. D. , Younghusband H. B.
Published: February 2005

Normal C57 Black mouse embryo cells did not form colonies in agarose, but rare variant (ar+) cells able to grow in agarose were detected. Fluctuation analysis showed that ar+ variants arose by spontaneous mutation in the cultured cells. The frequency of ar+ variants was increased by treating cells with N‐methyl‐N'nitro‐N‐nitrosoguanidine or ethyl methane sulphonate, or by abortive infection by human adenovirus type 5. Induced ar+ cells were fibroblastic; most grew slowly and had slightly reduced saturation density and increased serum requirement, but formed colonies in agarose. Fourteen of twenty ar+ clones induced by Ad5 were T antigen negative and two of these were also negative when tested for viral DNA. Six clones contained a few cells that were T antigen positive when first tested, but were negative when retested later. The ar+ variants were tumorigenic in athymic and in normal syngeneic mice. The results suggest that the ar+ phenotype can arise by spontaneous or chemically‐induced mutation, and can be induced by adenovirus by a process different from classical transformation.
The relationship of ornithine decarboxylase activity to proliferation and differentiation of L6 muscle cells
Authors: Stoscheck Christa M. , Florini James R. , Richman Robert A.
Published: February 2005

Four different approaches were used to test a postulated relationship between ornithine decarboxylase (ODC) activity and L6 muscle cell proliferation. In the first approach, the level of ODC activity in myoblasts was compared to that of myotubes. The fusion of proliferating myoblasts to form postmitotic myotubes is a unique property of skeletal muscle differentiation which offers special advantages for this study. If ODC activity reflects proliferation, it should be reduced in myotubes. Four hours after the addition of 10% horse serum (HS), ODC activity in myoblasts was 140 times that in myotubes. The small amount of ODC activity measured in myotube cultures could be attributed to remaining myoblasts. In the second approach, cell density was varied to alter the rate of cell division. Density‐dependent inhibition of myoblast proliferation was closely correlated with a decrease of ODC activity. In the third approach, HS concentration was varied to alter the rate of cell division. Through a tenfold change of HS concentration, ODC activity paralleled cell proliferation. In the fourth approach, 1mM putrescine or spermidine was added with 10% HS to myoblasts. Although these treatments prevented the increase in ODC in response to HS, cell division was unimpaired; it seems likely that the polyamines entered the cell and substituted for endogenous polyamines. Mixing experiments indicated the presence of an inhibitor, tentatively identified as ODC antizyme, in homogenates of polyamine‐treated cells. We conclude that ODC activity closely reflects proliferation in muscle cells, although the presence of extracellular polyamines may disrupt this association.
Isolation and charactrization of a morphologic variant of chinese hamster (CHO) cells
Authors: Sisskin Enid E. , Weinstein I. Bernard
Published: February 2005

An epithelioid clone of Chinese hamster (CHO) cells which is spontaneously transformed was exposed to the mutagen N‐methyl‐N‐Nitro‐N‐Nitrosoguanidine (MNNG), and a fibroblastic variant, clone CHO‐F2, was isolated. This clone is partially reverted in several of the in vitro properties characteristic of transformed cells. When compared to wild type CHO, CHO‐F2 has a longer doubling time, a lower saturation density and less piling up at high cell density, and a higher serum requirement. CHO‐F2 also elaborates less plasminogen activator and has more abundant microtubules and actin cables. On the other hand, both CHO and CHO‐F2 grow in agar suspension (although CHO‐F2 grows with a lower efficiency), both lack detectable LETS protein, and both are tumorigenic in nude mice. Thus, expression of the individual properties frequently associated with transformation and tumorigenicity can be dissociated.

The most critical biochemical change in CHO‐F2 appears to bean increase in the intracellular level of cyclic AMP, when compared to CHO, and several growth and morphological properties of CHO‐F2 resemble those induced in wild type CHO exposed to exogenous dibutyryl cyclic AMP. Therole of cyclic AMP in expression of the transformed phenotype and the significance of individual in vitro parameters of transformation with respect to tumorigenicity are discussed.
The suppression of cellular proliferation in SV40‐transformed 3T3 cells by glucocorticoids
Authors: Young Delano V. , Dean Michael C. , Young Delano V.
Published: February 2005

Glucocorticosteroids, when added two hours after cell plating to SV40‐transformed, 3T3 mouse fibroblasts in low serum (0.3%v/v), biotin‐supplemented medium, suppress cellular proliferation by 24 hours. While some cell death probably occurs, the growth inhibition is not primarily due to cytotoxicity and cytolysis. This conclusion is supported by the following: (1) both dead and viable cell numbers are suppressed, (2) little cell debris is evident in the medium, and (3) very high concentrations of glucocorticoids do not cause an increase in the dead cell count. Furthermore, this growth suppression, which is specific for glucocorticoids since several non‐glucocorticoid steroids have no inhibitory effect, is not permanent nor irreversible. Removal of the glucocorticoid and replacement with 10% serum restore rapid proliferation.

Although higher concentrations (1% and 10%) of serum afford some protection against glucocorticoid inhibition, this protection is not simply a consequence of faster growth rates. SV3T3 cells can be grown in serum‐free medium supplemented with biotin, transferrin, insulin, and epidermal growth factor (EGF). Under these conditions growth rates are comparable to high serum media, yet glucocorticoids are still powerful inhibitors. However, the omission of insulin from serum‐free, glucocorticoid cultures does result in observable cell death and lysis.

Flow microfluorometry and autoradiographic studies have determined that glucocorticoid‐inhibited cells are partially blocked in G1. The proportions of S phase and G2 + M cells are greatly reduced with an accompanying accumulation of G1 cells. These results suggest that glucocorticoids regulate a biochemical step(s) in G1 which is critical for DNA initiation.
Cholera toxin and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells
Authors: Taylor‐Papadimitriou Joyce , Purkis Patricia , Fentiman Ian S.
Published: February 2005

The growth of human mammary epithelial cells is stimulated by cholera toxin and analogues of cyclic AMP, while the growth of breast derived fibroblasts is inhibited. These compounds have little effect on DNA synthesis in the absence of other mitogens but show a synergistic effect with serum and/or EGF. The results suggest that high intracellular levels of cyclic AMP in human mammary epithelial cells increase the growth response of the cell to mitogens.
Growth arrest of AKR‐2B cells maintained in the presence of epidermal growth factor or 12‐O‐tetradecanolyhorbol‐13 acetate: Evidence for two separate G1 arrest points
Authors: Moses Harold L. , Proper Jacqueline A. , Volkenant Mary E. , Swartzendruber Douglas E.
Published: February 2005

Nontransformed mouse embryo derived AKR‐2B cells stop growing in the G1 phase of the cell cycle at saturation density due to depletion of serum growth factors, whereas a chemically transformed derivative line (AKR‐MCA) arrests in G1 at a higher saturation density due to depletion of amino acids and glucose. Stimulation of DNA synthesis is inhibited in the AKR‐2B cells, but not in the AKR‐MCA cells, by two inhibitors of RNA metabolism, α‐amanitin and 5‐fluorouridine (5‐FU). To determine whether the AKR‐MCA cells growth arrest at a uniue point in G1 or whether they arrest in a physiologic state which can also be achieved by the nontransformed cells, AKR‐2B cells were maintained in medium with 10% serum containing the mitogens, epidermal growth factor (EGF) or 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), until they reached saturation density or were arrested at subconfluence by artificial deletion of amino acids from the medium. The AKR‐2B cells maintained in EGF or TPA stopped growing in G1 at a higher saturation density, due to depletion of amino acids. Cells arrested in EGF or TPA or in amino acid deficient medium had a shortened interval between stimulation and the onset of DNA synthesis, and the stimulation of DNA synthesis was not inhibited by α‐amanitin or 5‐FU. The data show that the nontransformed AKR‐2B cells have two different arrest states which may represent two separate and distinct G1 arrest points—a growth factor deficiency arrest point and a nutrient deficiency arrest point. The nutrient deficiency arrested cells were very similar to the G1 arrested transformed AKR‐MCA cells.
Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells
Authors: Karin Michael , Herschman Harvey R.
Published: February 2005

HeLa cells grown in chemically defined medium lacking glucocorticoids synthesize metallothioneins, low molecular‐weight heavy‐metal binding proteins. Dexamethasone and hydrocortisone increase the rate of metal‐lothionein synthesis five‐ to ten‐fold. Maximal induction is achieved with 10–8M dexamethasone and 10–7M hydrocortisone. Half‐maximal induction is achieved at 5 ± 10–9M dexamethasone and 5 ± 10–8M hydrocortisone. Although carried for many generations in the absence of any glucocorticoids, HeLa cells (clone S) contain 25,000 specific 3H‐dexamethasone receptors that translocate into the nucleus after one hour of incubation. 3H‐dexamethasone binds to a single class of receptors with an apparent Kd = 18.8 nM. A variety of steroids can be classified into three classes, based on their effect on metallothionein synthesis: (a) full agonists (optimal inducers), (b) intermediate effectors which have either partial agonist or antagonist activities, and (c) inactive steroids. There is a correlation between the effects on metallothionein synthesis of different steroids and their ability to compete with 3H‐dexamethasone binding. We conclude that metallothionein is induced in HeLa cells by a glucocorticoid receptor mediated mechanism.
Effects of insulin on glycogen metabolism of serum‐deprived BHK‐21 fibroblasts
Authors: Lavis Victor R. , Thompson W. Joseph , Strada Samuel J.
Published: February 2005

We have studied the effects of insulin on glycogen metabolism of cultured BHK‐21 fibroblasts. Addition of insulin to cells cultured in 0.5% serum stimulated incorporation of 5mM 14C‐glucose into glycogen and increased cellular glycogen content, without inducing proliferation. With serum‐deprived cells incubated for 2 hr, maximal stimulation of incorporation of glucose into glycogen was 3.5‐fold and the half‐maximal dose of insulin was 5nM. After culture for 24 hr with 100nM insulin, incorporation was increased 13‐fold and glycogen content was increased by 44%. Incorporation of glucose into glycogen was reduced by agents which elevate cellular adenosine 3,5‐cyclic monophosphate (cyclic AMP): 10μM prostaglandin E1 (PGE1), 0.25 mM 1‐methyl‐3‐isobutylxanthine (MIX), 10μM L‐epinephrine (L‐EPI), and 10μM L‐isoproterenol (L‐ISO). Culture with 100nM insulin for 2 hr stimulated incorporation three‐fold in the presence of any of these compounds, but insulin did not affect cellular cyclic AMP.

At all times from 1 hr to 24 hr after addition of insulin to serum‐deprived cultures, the 1‐min uptake of 10μM 3H‐2‐deoxyglucose (3H‐2DG) was increased. There were small (30 to 40%) increases in glycogen synthase I activity at 15 and 30 min but not 4 hr after addition of insulin. After 24 hr with insulin, total glycogen synthase and phosphorylase activities were increased approximately two‐fold, without changes in their activation states.

We conclude that insulin promotes glycogenesis in serum‐deprived BHK‐21 cells. This response is mediated principally by increased entry of glucose into cells, and is not mediated by a change in cellular cyclic AMP concentration.
The effect of serum and multiplication stimulating activity on L6 myoblast growth: The lack of correlation with cyclic nucleotide changes
Authors: Richman Robert A. , Weiss James P. , Roberts Susan B. , Florini James R.
Published: February 2005

The role of cAMP and cGMP in triggering proliferation of rat myoblasts was evaluated by: (1) measuring effects of mitogens on intracellular cyclic nucleotide levels, and (2) observing effects of agents which altered cyclic nucleotide levels on cell proliferation. Multiplication stimulating activity (MSA, 1 μg/ml), a member of the somatomedin family, stimulated cell proliferation after 48 hr. It had little effect on cellular cyclic nucleotide levels, measured by radioimmunoassay. Horse serum (HS) and fetal bovine serum (FBS) stimulated cell proliferation approximately equally. Neither affected cAMP levels; FBS reduced cGMP to 33% of control values, but HS had no effect. Thus, there was no simple correlation between mitogenic action and cyclic nucleotide levels at any time from 5 min to 24 hr after addition of a purified mitogen or serum. Furthermore, agents which caused substantial changes in cyclic nucleotide levels had no effect on cell proliferation. Prostaglandin E1 (5 μM) elevated cAMP 440% without affecting cGMP levels or cell growth. A potent phosphodiesterase inhibitor, 1‐methyl‐3‐isobutylxanthine (MIX, 0.1 mM), when added alone or in the presence of MSA, HS, or FBS, elevated cAMP 200% and cGMP 167%, but it had little effect on their mitogenic action. Lastly, a purified mitogen such as MSA, unlike serum, must be present for extended periods of time in order to stimulate cell proliferation. This makes it unlikely that a trigger mechanism functions in initiating cell division. We conclude that neither cAMP nor cGMP appear to be second messengers for the mitogenic action of MSA or serum on muscle cells.
Nucleic acids methylation of synchronized BHK 21 HS 5 fibroblasts during the mitotic phase
Authors: Bugler B. , Bertaux O. , Valencia R.
Published: February 2005

The methylation of nucleic acids has been investigated during the cell cycle of an asparagine dependent strain of transformed fibroblasts (BHK 21 HS 5). The synchrony was carried out by a partial asparagine starvation of cells for 24 hours. The amino acid supply induced all cells to enter synchronously the G1 phase. Methylation and DNA synthesis were respectively measured by pulsed [methyl‐14C] methionine and [methyl‐3H] thymidine incorporation. DNA methylation followed a biphasic pattern with maximal methyl incorporations during both S phase and mitosis.

A partial desynchronisation induced the S phase of the second cycle to proceed before all the cells have achieved their division. Hydroxyurea was used in order to inhibit the DNA synthesis of cells entering the second cell cycle, which might interfer with the mitosis of the first one. The inhibitor was added either at the first beginning of cell division or during all the G1 phase. In both conditions it suppressed 3H thymidine incorporation of the second cycle. However, mitosis took place and methylations occurred as in previous experiments.

The DNA methylation of the mitotic phase in the first cell cycle could thus be dissociated from the classical post‐synthetic DNA maturation and did not correspond to any DNA methylation appearing in the course of the second cell cycle.
The energy charge in wild‐type and respiration‐deficient chinese hamster cell mutants
Authors: Soderberg K. , Nissinen E. , Bakay B. , Scheffler I. E.
Published: February 2005

High pressure liquid chromatography was used to determine the base, nucleoside, and nucleotide levels in wild type and a series of respiration‐deficient Chinese hamster cell mutants. From this analysis the size of the total adenylate pool and the energy charge could be calculated for each cell line. We find a constant energy charge, as expected, but the adenylate pool seems to be considerably lower in the respiration‐deficient mutants.
In vitro proliferation and lifespan of bovine aorta endothelial cells: Effect of culture conditions and fibroblast growth factor
Authors: Duthu Gwen S. , Smith James R.
Published: February 2005

The effect of culture conditions on calf dorsal aorta endothelial cells was studied. Population doubling time varied as a function of the cell seeding density, growth medium, serum supplement, and concentration of fibroblast growth factor (FGF). The shortest population doubling time was found for cells (population doubling level 0–30) grown in Eagle's Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 100 ng/ml FGF. The stimulatory effect of FGF on bovine endothelial cell proliferation was dependent on cell inoculation density. FGF significantly increased cell division rate at cell inocula less than 1 ± 104 cells/cm2 but not at higher densities. The population doubling time and cell size increased as the mass culture population doubling level increased. The replicative lifespans of bovine endothelial cells grown in medium supplemented with 20% FBS were 10–15% greater than parallel cultures supplemented with 10% FBS. Cultures grown in medium supplemented with 10% FBS and 50 ng/ml FGF showed a 50% increase in replicative lifespan compared to cultures grown in medium supplemented with 10% FBS alone. When FGF was used the increase in the number of doublings was a function of the length of time the cells were grown in the presence of FGF. This report extends comparable observations on the in vitro aging of human diploid fibroblasts to bovine endothelial cells.
Inhibition of the adipose conversion of BALB/c 3T3 cells by 12‐0‐tetradecanoylphorbol‐13‐acetate: Dependence on pH reduction via lactic acid production
Authors: O'Brien Thomas G. , Saladik Douglas
Published: February 2005

The effect of modifying culture pH on the adipose convesion of BALB/c 3T3 cells was studied. We have previously shown that the tumor promoter 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) stimulates cellular lactic acid production, lowers the pH of the culture medium, and inhibits differentiation in this system. We now report that addition or organic acids such as D‐ or L‐lactic acid or acetic acid to the culture medium mimicked the effect of TPA by lowering pH, stimulating cellular lactic acid production, and inhibiting the adipose conversion. Lowering the pH by changing the NaHCO3 concentration also inhibited differentiation.

A comparison of Dulbecco's modified minimal essential medium (DMEM) vs minimal essential medium (MEM) indicated that in the former media, the rate and extent of differentiation was greater than in the MEM, and TPA neither inhibited differentiation nor stimulated lactate production. Our standard MEM medium could be made similar to DMEM with respect to the cells' response to TPA simply by raising the NaHCO3 concentration, hence, the buffering capacity, of MEM to that present in DMEM, suggesting that TPA can only inhibit differentiation in this system if it can lower culture pH via lactate production.
A novel synergistic effect of alanosine and guanine on adenine nucleotide synthesis in mammalian cells. Alanosine as a useful probe for investigating purine nucleotide metabolism
Authors: Gupta Radhey S.
Published: February 2005

A novel synergistic effect of the antitumor agent alanosine (2‐amino‐3‐(hydroxynitrosoamino) propionic acid), which specifically inhibits the enzyme adenylosuccinate synthetase (ASS) and guanine on the growth of Chinese hamster ovary (CHO) cells and human diploid fibroblasts (HDF) has been observed. In the presence of subinhibitory concentrations of alanosine, both CHO cells and the HDF show excessive sensitivity to exogenous guanine—a phenotype which closely resembles that seen with some of the mutants containing reduced enzymatic activity of ASS. The growth inhibitory effects of alanosine, or alanosine and guanine, on CHO cells are completely reverted by the addition of adenine to the culture medium, and the synergistic effect of guanine is not observed in mutants which lack the enzyme hypoxanthine‐guanine phosphoribosyl transferase. These results suggest that guanine nucleotides exert a regulatory effect on the activity of the enzyme adenylosuccinate synthetase. The ability to confer the guaninesensitive phenotype and its modulation by subinhibitory concentrations of alanosine in different cell types indicates that alanosine provides a useful probe for investigating the regulation of purine nucleotide metabolism in mammalian cells.
Some metabolic differences between human skin and aponeurosis fibroblasts in culutre
Authors: Lemonnier F. , Gautier M. , Wolfrom C. , Lemonnier A.
Published: February 2005

Two types of human fibroblast strains were studied in culture. One was derived from abdomen skin and the other from abdominal muscle aponeurosis. Tissue‐specific differences were found between thése two cell strains. Skin fibroblasts had faster doubling time, smaller cell volume, and lower glucose consumption when compared to aponeurosis fibroblasts. Furthermore, extracellular amino acid variations showed some specific differences, in particular a lack of serine consumption in skin fibroblasts.