bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of revertant cell lines. VII. DNA synthesis and mitotic rate of serum‐sensitive revertants in non‐permissive growth conditions
Authors: Vogel Arthur , Pollack Robert
Published: February 2005

The ability to synthesize DNA and enter mitosis was studied in Balb/c and Swiss 3T3 cells, SV40 and MSV‐transformed 3T3 cells and revertants of these transformed cells in cultures of different serum concentrations and cell densities. Three ways were found by which cells were able to maintain a constant cell number in non‐permissive growth conditions: cessation of DNA synthesis, synthesis of DNA coupled with failure to enter mitosis, and the slow traverse of the cell cycle coupled with cell shedding.

Growth control of the revertant of an MSV‐transformed Balb/3T3 cell most closely resembled that of Balb or Swiss 3T3. This line did not grow in 1% serum and did not synthesize DNA in either non‐permissive condition.

Serum‐sensitive revertants of SV40‐transformed 3T3 cells are also unable to grow in 1% serum and also do not grow beyond confluence in 10% serum, but these cells differ from 3T3 in the manner in which this growth arrest is accomplished. In 1% serum, revertants synthesize DNA but do not enter mitosis. At confluence in 10% serum, they slowly traverse the cell cycle, with dividing cells replacing cells that are shed into the medium.
Neoplastic differentiation: Interaction of simian virus 40 and polyoma virus with murine teratocarcinoma cells in vitro
Authors: Swartzendruber D. E. , Lehman J. M.
Published: February 2005

The host‐virus interactions of Simian virus 40 (SV40) and polyoma virus (Py) with cell lines established from a teratocarcinoma were studied. The cells utilized in this study were the multipotential stem cell of the teratocarcinoma, embryonal carcinoma, and differentiated cells derived from embryonal carcinoma. Several lines of differentiated cells were established in vitro which included parietal yolk sac, epithelial, and spindle cell types. Embryonal carcinoma cells are not susceptible to infection by either SV40 or Py virus. However, differentiated cells are susceptible to infection by these viruses. The differentiated cells are permissive for Py virus replication and nonpermissive for SV40. Several continuously growing cell lines have been established from the SV40 infected cultures which express T antigen in 100% of the cells. The results indicate that undifferentiated embryonal carcinoma cells and their differentiated progeny respond quite differently to challenge with these two oncogenic DNA viruses.
The early effects of ouabain on potassium metabolism and rate of proliferation of mouse lymphoblasts
Authors: Cuff Joyce M. , Lichtman Marshall A.
Published: February 2005

Murine lymphoblasts grown in suspension culture in the presence of ouabain showed a dose dependent and sequential decrease in 86Rb+ (K+ analogue) influx, cellular potassium content, and growth rate. An increase in eosin staining and a decrease in cell number was observed after two hours in the presence of 1 mM ouabain; 1 μM ouabain was without effect on any of the parameters measured. Ouabain inhibition was rapidly and completely reversible at concentrations that were not cytotoxic.
Somatic genetic analysis of cyclic AMP action: Selection of unresponsive mutants
Authors: Coffino Philip , Bourne Henry R. , Tomkins Gordon M.
Published: February 2005

Dibutyryl cyclic AMP and theophylline kill S49.1 mouse lymphoma tissue culture cells. When cells are grown in soft agar with these drugs, the few clones that survive are resistant to cytolysis. The rate of mutation to resistance is 1–3 × 10−7/cell/generation in both diploid and tetraploid cells. The incidence of mutants is increased by treatment with a chemical mutagen ICR 191. The mutation is consistently associated with greatly reduced or absent cytoplasmic cyclic AMP binding protein. These results suggest that a somatic mutation leads to a defect of the protein kinase regulatory subunit and that activity of this kinase is required for induction of cell death by cyclic AMP.
Effect of ploidy on spontaneous mutation rate to asparagine non‐requirement in cultured Cells
Authors: Prickett Mary Sue , Coultrip Laura , Patterson M. K. , Morrow John
Published: February 2005

Variations in ploidy do not affect the spontaneous mutation rate to asparagine non‐requirement in Jensen rat sarcoma cells cultivated in vitro.
Control of normal differentiation of myeloid leukemic cells. VIII. Induction of differentiation to mature granulocytes in mass culture
Authors: Fibach Eitan , Sachs Leo
Published: February 2005

There are three types of myeloid leukemic cells, IR+D+, IR+D− and IR−D−. IR+D+ cells were induced to differentiate to granulocytes in mass culture in liquid medium by conditioned medium (CM) from cultures of lungs from mice injected with endotoxin. About 90% of the leukemic cells were induced to differentiate, 50% to mature granulocytes and 40% to intermediate stages. An efficient induction of granulocyte differentiation was also obtained with CM from primary cultures of rat embryo or human spleen and there was a lower activity with CM from various other sources. IR+D− cells were induced to differentiate to about 20% cells with intermediate stages but not to mature granulocytes; IR−D− cells could not be induced to differentiate to intermediate or mature stages. IR+D+ cells were induced to form intermediate stages of granulocyte differentiation, to phagocytose and to attach to the surface of the Petri dish, three days after incubation with CM. Optimum induction of mature granulocytes required six more days incubation with CM. Mature granulocytes induced from leukemic cells showed cytochemical properties and a morphology in the electron microscope similar to that of normal mature granulocytes. These induced granulocytes did not form leukemiac in animals or colonies in agar. The granulocytes induced from the myeloid leukemic cells, therefore, behaved like normal mature granulocytes.
Separation of subpopulations of in vitro colony forming cells from mouse marrow by equilibrium density centrifugation
Authors: Williams Neil , Van Engh Gerrit J. Den
Published: February 2005

Equilibrium density centrifugation was used to characterise and separate subpopulations of mouse haemopoietic progenitor cells capable of producing colonies of granulocytes and macrophages in vitro. The material used to induce colony formation (CSF) was prepared from an extract of pregnant mouse uteri. This CSF preparation was found to be free of factors modifying the response. Under these culture conditions, in vitro colony forming cells (CFU‐c) were found to be relatively homogeneous in their buoyant density. This homogeneity was independent of CSF concentration. A heterogeneous density profile of CFU‐c was obtained when various cell fractions were cultured in the presence of CSF and rat blood lysate. The majority of the additional cells which responded to erythrocyte lysate were dense (modal density 1.080 g/cm3) compared to CFU‐c which respond to CSF alone (modal density 1.074 g/cm3). It is concluded that in vitro colonies induced by CSF and in vitro colonies grown in the presence of CSF and erythrocyte lysate reflect two different populations of CFU‐c.
Isolation and characterization of alkaline phosphatase‐constitutive variants from chinese hamster ovary cells (CHO‐K1)
Authors: Nose Kiyoshi , Katsuta Hajim
Published: February 2005

Pure clones with high alkaline phosphatase (ALP) activity were isolated from Chinese hamster ovary (CHO‐K1) cells which showed no detectable ALP activity. ALP‐positive cells appeared at the frequency of 10−4 in the N‐methyl‐N'‐nitro‐N‐nitrosoguanidine‐treated cell population.

With respect for cellular morphology, plating efficiency and several other enzymatic activities, no distinct difference was found between the original CHO‐K1 cells and its ALP‐positive variants, although some alterations in karyotype were found.

Levels of ALP activity in these clones was stably maintained during serial cultivation. Some of the enzymological properties of ALP in the isolated clones were similar to those in bone or kidney.
Autoradiographic studies of nicotinic acid utilization in human‐mouse heterokaryons and inhibition of utilization in newly‐formed hybrid cells
Authors: Rechsteine Martin , Hill Kim R.
Published: February 2005

Although most mammalian cell lines can utilize either nicotinic acid or nicotinamide for the biosynthesis of nicotinamide adenine dinucleotide (NAD), thymidine kinase‐deficient, mouse 3T3–4F cells are unable to utilize nicotinic acid. When 3T3–4E cells were fused with human D98/AH2 cells, autoradiography showed that the resultant heterokaryons synthesized NAD from nicotinic acid at rates comparable to the human parental cell. The rate of nicotinic acid utilization in heterokaryons remained unchanged over the fourday period of study following cell fusion. In contrast to the results observed with heterokaryons, nicotinic acid utilization was markedly reduced in hybrid cells. Of 100 hybrid clones examined at four or five days following cell fusion, 60 utilized nicotinic acid at rates less than one tenth that of the parental human cell. Similar results were observed in hybrid clones at nine or ten days following fusion. Uniformly high rates of NAD biosynthesis were observed in hybrid clones with nicotinamide as the precursor. This excludes the possibility that the reduction in nicotinic acid utilization in hybrid cells is due to a general metabolic dysfunction. The biochemical mechanism by which nicotinic acid utilization is markedly reduced has not been determined with certainty, however, several observations suggest genetic suppression.
Stimulation of lactic acid production in chick embryo fibroblasts by serum and high pH in the absence of external glucose
Authors: Fodge D. W. , Rubin H.
Published: February 2005

Lactic acid production by chick embryo fibroblasts occurs in the absence of exogenous glucose. Fifteen to 50‐fold less lactic acid is formed in the absence of glucose than in its presence. Nevertheless, serum and pH stimulation enhances this residual lactic acid production to the same relative extent as when glucose is present. The amount of lactic acid formed cannot be accounted for by the catabolism of residual glucose in the medium since its concentration is less than one‐tenth that of the lactic acid eventually produced. Moreover, the residual glucose concentration remains constant or increases during the course of the experiment. To a large extent lactic acid accumulation in the absence of external glucose is dependent on the presence of amino acids in the medium, but amino acid transport is not affected by the stimulatory agents used in this study. The results suggest that treatments which stimulate cell multiplication also activate those enzymatic pathways which convert amino acids to pyruvic and thence to lactic acid.
The control of liver regeneration by parathyroid hormone and Calcium
Authors: Rixon R. H. , Whitfield J. F.
Published: February 2005

Following partial hepatectomy in rats, there were two bursts of hepatocyte DNA‐synthetic and mitotic activity which were produced by two subpopulations having different rates of (nearly synchronous) proliferative development. Only about 50% of the cells in both subpopulations could initiate DNA synthesis and enter mitosis when exposed to the hypocalcemic conditions in the parathyroprivic rat for 24 hours before partial hepatectomy. The proliferatively incompetent hepatocytes in these hypocalcemic rats could be induced to initiate their DNA synthetic and mitotic activity by an intraperitoneal injection of the calcium‐mobilizing parathyroid hormone (50 USP units/100 g) as late as 12 hours after partial hepatectomy. Single intraperitoneal injections of calcium (0.25 mg/100 g) could also restore the proliferative competence of these hepatocytes, but only when injected at specific periods following partial hepatectomy. The injection of calcium 12 to 15 hours after partial hepatectomy induced hepatocytes in the first subpopulation to finish their development and enter mitosis, but did not affect the second, more slowly developing, subpopulation. Calcium had to be injected 25 hours after partial hepatectomy to stimulate proliferation in this second subpopulation. These data suggest that the hepatocytes which became proliferatively incompetent by prolonged exposure to a hypocalcemic environment are proliferatively activated by partial hepatectomy, but their proliferative development stops at a calcium‐dependent stage near the end of the pre‐replicative phase of development.
Maturation of membrane function: Transport of amino acid by rat erythroid cells
Authors: Wise W. C.
Published: February 2005

The membrane changes which occur during cellular maturation of erythroid cells have been investigated. The transport of α‐aminoisobutyric acid, alanine, and N‐methylated‐α‐aminoisobutyric acid have been studied in the erythroblastic leukemic cell, the reticulocyte, and the erythrocyte of the Long‐Evans rat. The dependence of amino acid transport on extracellular sodium concentration was investigated.

Erythrocytes were found to transport these amino acids only by Na‐independent systems. The steady state distribution ratio was less than 1. Reticulocytes were found to transport α‐aminoisobutyric acid and alanine by Na‐dependent systems, but only small amounts of N‐methylated‐α‐aminoisobutyric acid. Small amounts of these amino acids were transported by Na‐independent systems. The steady state distribution ratio was greater than one for Na‐dependent transport. The erythroblastic leukemia cell, a model immature erythroid cell, showed marked Na‐dependence (>90%) for α‐aminoisobutyric acid and alanine transport, and >80% for the Na‐dependent transport of N‐methyl‐α‐aminoisobutyric acid. The steady state distribution ratio for the Na‐dependent transport was >4.

In the erythroblastic leukemic cell, at least three Na‐dependent systems are present: one includes alanine and α‐aminoisobutyric acid, but excludes N‐methyl‐α‐aminoisobutyric acid; one is for α‐aminoisobutyric acid, alanine and also N‐methyl‐α‐aminoisobutyric acid; and one is for N‐methyl‐α‐aminoisobutyric acid alone. In the reticulocyte, the number of Na‐dependent systems are reduced to two: one for α‐aminoisobutyric acid and alanine; one for N‐methyl‐α‐aminoisobutyric acid. In the erythrocytes, no Na‐dependent transport was found. Therefore, maturation of the blast cell to the mature erythrocyte is characterized by a systematic loss in the specificity and number of transport systems for amino acids.
Exodus of 42K+ and 86Rb+ from rat thymic and human blood lymphocytes exposed to phytohemagglutinin
Authors: Segel George B. , Gordon Bruce R. , Lichtman Marshall A. , Hollander Marsha M. , Klemperer Martin R.
Published: February 2005

We have found that PHA produces an alteration in the lymphocyte membrane which allows 86Rb+ or 42K+ in prelabeled lymphocytes to exchange for cations present in washing solutions. These observations suggested that PHA might induce an increase in the exodus of intracellular potassium during incubation in physiologic media. We, therefore, examined 86Rb+ and 42K+ efflux from rat and human lymphocytes during incubation in tissue culture medium. The rate constant for efflux, Ke, was significantly increased by PHA.86Rb+ efflux was increased by 27% in rat thymic lymphocytes and by 78% in human blood lymphocytes following PHA treatment.
Bromodeoxyuridine induced variations in the level of alkaline phosphatase in several human heteroploid cell lines
Authors: Bulmer Deborah , Stocco Douglas , Morrow John
Published: February 2005

The level of alkaline phosphatase in a number of established cell lines of human origin can be modified by exposure to non‐lethal concentrations of bromodeoxyuridine (BRdU). In the several cell lines examined an inverse relationship between amount of induction and constitutive level of the enzyme was observed. Thus, the H.Ep 2 line, which had the highest basal level of enzyme, was reversibly repressed following exposure to the drug, whereas other cell lines with relatively low constitutive enzyme levels were induced to a maximum of 10‐fold following exposure. Initiation of induction required from 24 to 48 hours, and as short an exposure (“pulse”) as five hours was sufficient to produce induction.

Exposure to visible light had no effect upon the repression of alkaline phosphatase in H.Ep 2 by BRdU. Induction did not occur in non‐dividing, serum starved cells. The time course of induction by BRdU and hydrocortisone was similar, and simultaneous exposure of the cells to both agents resulted in no greater induction than that observed with either drug alone.

Experiments utilizing mitomycin C yielded significant induction in the presence of this agent alone, and somewhat less induction when both mitomycin C and BRdU were added simultaneously. These results suggest that DNA synthesis is required for BRdU‐mediated induction of alkaline phosphatase.
Substrate‐attached serum and cell proteins in adhesion of mouse fibroblasts
Authors: Culp Lloyd A. , Buniel Josefina F.
Published: February 2005

The effects of serum and coatings of substrate‐attached material (SAM, which remains tightly adherent to the substrate after EGTA‐mediated removal of cells) on the kinetics of attachment of DNA‐radiolabeled BALB/c 3T3, SV40‐transformed 3T3, and concanavalin A‐selected revertant cells to glass coverslips were studied. The presence of serum in the medium of attaching cells had a marked effect on (1) the initial time lag before stable attachment of cells, (2) the maximum level of attached cells, (3) the stability of attachment, and (4) pseudopodial spread of the cell over the substrate. These serum effects could be mimicked by measuring attachment in medium without serum and with use of serum‐preadsorbed or 3T3 SAM‐coated coverslips. Enzymatic treatment of serum‐preadsorbed substrates indicated that the factor(s) in serum which affects attachment is very trypsin‐sensitive. Serum preadsorption of substrates stimulated attachment of SVT2 cells in medium with serum in a manner very similar to the effects of 3T3 SAM coatings, while attachment of 3T3 or revertant cells was unaffected.

Slab gel electrophoretic analysis (PAGE‐SDS gels) identified eight major serum proteins by Coomassie blue staining (a) which bind to the substrate in the absence of cells and (b) which persist on the substrate after growth to confluence of 3T3 or SVT2 cells; this suggests that major breakdown of serum‐adsorbed components does not occur during growth of normal or transformed cells. Seven radioactive SAM proteins were detected by autoradiography in 3T3 or SVT2 SAM electropherograms — two of which are high molecular weight components which correspond to the glucosamine‐radiolabeled hyaluronate proteoglycans observed previously; the remaining five are newly‐identified proteins in SAM (one of these proteins appears to be actin). 3T3 and SVT2 cells have unique proportions of these seven components.

The data are consistent with the idea that normal or virus‐transformed cells do not attach directly to the culture substrate, but to specific classes of substrate‐adsorbed serum proteins via deposition of specific classes of cell surface proteins and polysaccharides.
Synthesis of immunoglobulin by substrate attached mouse myeloma cells
Authors: Bargellesi Antonio , Damiani Guido , Kuehl W. Michael , Scharff Matthew D.
Published: February 2005

Cultured mouse myeloma cells grow in suspension and synthesize and secrete large amounts of immunoglobulin. Mouse myeloma cells which attach to a plastic substratum have been obtained by mutagenesis and subsequent selection. Normal mouse myeloma cells will also attach to plastic tissue culture dishes pre‐treated with poly‐L‐lysine. The attached cells synthesize and secrete the same large amounts of immunoglobulin as the suspended cells.
The role of calcium in the inhibition of cAMP accumulation in epiphyseal cartilage cells exposed to physiological pressure
Authors: Bourret L. A. , Rodan G. A.
Published: February 2005

A hydrostatic pressure of 60g/cm sq (0.85 psi) inhibits the accumulation of cAMP in cells isolated from the proliferative zone of chick‐tibia epiphyseal cartilage. The following findings indicate that this effect is mediated by a translocation of calcium: (i) the pressure enhances the cellular uptake of radio‐calcium; (ii) the pressure effect on cAMP can be simulated by the calcium‐ionophore A23187; (iii) the effects of pressure and A23187 are non‐additive; (iv) the pressure effect is not produced in the presence of ethylenebis‐(oxyethylenenitrilo)‐tetraacetic‐acid (EGTA); (v) the particulate adenyl cyclase activity of the proliferative zone is susceptible to non‐competitive calcium inhibition.

Throughout this study cells from the hypertrophic zone of the same epiphyses were used as controls. In these cells the calcium uptake was enhanced by pressure, but the cAMP level was not affected by pressure, A23187 or EGTA. This change in responsiveness, which accompanies the maturation of the cartilage cells, was shown to be due to a decrease in the calcium‐inhibition of adenylate cyclase.
Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A
Authors: Iversen Jens‐Gustav
Published: February 2005

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38°C, or the cellular uptake of isotope during a similar incubation was measured.

The influx rate of untreated thymocytes was: 2.3·10−12 moles cm −2. S−1 and efflux rate: 1.8·10−12 moles cm −2· S−1·

When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 μg/ml, but concentrations as low as 0.75 μg/ml were effective.

Hydrocortisone resistant thymocytes responded at least as well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times.

Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl−: 43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present.

Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A.

The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, α‐methyl‐D‐mannoside.
Ascorbate‐independent proline hydroxylation resulting from viral transformation of balb 3T3 cells and unaffected by dibutyryl camp treatment
Authors: Evans Carla A. , Peterkofsky Beverly
Published: February 2005

Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact‐inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki‐3T3; and dibutyryl cAMP (dbcAMP)‐treated Ki‐3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85–90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2–3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki‐3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki‐3T3 or dbcAMP‐treated Ki‐3T3 cell cultures either in logarithmic phase or stationary phase. Ki‐3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C‐glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascorbate‐in‐dependent hydroxylation was altered by treatment with dbcAMP.
Lysosomal hydrolase secretion by Tetrahymena: A comparison of several intralysosomal enzymes with the isoenzymes released into the medium
Authors: Blum J. J.
Published: February 2005

Tetrahymena pyriformis were grown in proteose‐peptone medium and then washed and incubated in a dilute salt solution for one hour. The cells were then discarded and the lysosomal hydrolases that had been secreted were subjected to DEAE cellulose column chromatography. At least three iso‐enzymes of acid phosphatase, three of acid protease, and two of β‐N‐acetylhexoseaminidase were found, as well as single peaks of α‐mannosidase, β‐galactosidase, and β‐fucosidase. The latter two activities were not resolved by the DEAE column and could not be separated in a second chromatographic step on CM‐cellulose.

Cells were also grown under identical conditions and homogenized in 0.25 M sucrose in order to allow comparison of some of the intracellular lysosomal hydrolases with their secreted counterparts. Two lysosomal populations were resolved by sucrose density gradient sedimentation, a heavy lysosomal fraction, centered at a density of about 1.25 gm/cm3, and a light lysosomal fraction, centered at a density of about 1.16 gm/cm3. These two populations differed in that the light lysosomes did not appear to contain significant amounts of β‐fucosidase, β‐galactosidase, or acid protease, whereas all six of the hydrolase activities studied were present in the heavy lysosomes. The light lysosomal peak occurred in cells grown to transition phase, but was markedly reduced in cells from cultures grown to stationary phase. In addition to these two fractions a third very light particle, containing only α‐mannosidase activity, was detected just inside the gradient.

Measurements were made of the effect of heat (10 minutes at 66°) and of a change in pH from 4.5 (standard assay condition) to 6.0 on the three acid phosphatases and two β‐N‐acetylhexoseaminidase isoenzymes resolved by DEAE column chromatography of the secreted hydrolases and on these hydrolyases in the heavy and light lysosomal fractions on the sucrose gradient. Use of the thermostability and pH criteria permitted computation of the expected properties of the intralysosomal acid phosphatase and hexoseaminidase activities if these consisted of the respective isoenzymes in the proportions secreted. It was found that neither the intralysosomal acid phosphatase nor the intralysosomal hexoseaminidase had the properties expected if they consisted of the secreted mixture of the respective isoenzymes, indicating that modification of some of these isoenzymes may have occurred during the 1‐hour starvation period or after secretion.
Derepression and carrier turnover: Evidence for two distinct mechanisms of hexose transport regulation in animal cells
Authors: Christopher C. William , Colby Wendy W. , Ullrey Donna
Published: February 2005

Hexose uptake by hamster cells was increased five to ten fold by either substituting D‐fructose for glucose or by completely omitting D‐glucose from the culture medium for 24 to 48 hours. Conversely, when cycloheximide was present for 24 hours in media containing glucose, up to 20‐fold decreases in hexose uptake were observed. However, these decreases in uptake activity were only observed over a narrow range of cycloheximide concentrations. After extended exposure to low concentrations of cycloheximide (0.05 to 10 μg/ml), the uptake by the fed cells decreased parallel with inhibition of protein synthesis whereas at high concentrations (>50 μg/ml) uptake was increased. Cells deprived of glucose and maintained in the presence of cycloheximide did not show decreases in uptake activity. In separate experiments the high uptake rates of glucose‐starved cells could be decreased by addition of glucose to the glucose‐free medium. The reversal was complete in 6 to 8 hours. The analog of glucose, 2‐deoxy‐D‐glucose, did not promote the time‐dependent decrease suggesting that the 6‐phosphoester of glucose is not an inhibitor of transport. In addition, when cycloheximide is added at the same time as glucose, there is no decrease in uptake for at least 12 hours. We propose that turnover of components of hexose uptake systems could account for part of the control of hexose transport. Moreover, the results indicate that the turnover mechanism becomes inactive during glucose starvation and must be resynthesized following refeeding of the starved cells with glucose.
Why do tumor cells have a high aerobic glycolysis?
Authors: Racker Efraim
Published: February 2005

In Ehrlich ascites cells and several other tumors, the high aerobic glycolysis is maintained by generation of ADP and Pi by the plasma membrane Na+K+ ATPase. The high ATPase activity is caused by a defective pump that operates at a low efficiency. Studies of the mechanism of action of the Na+K+ ATPase and other pump ATPases suggest several alternative mechanisms that might account for the decreased efficiency. The possibility of involvement of a proteolipid is under investigation.
Studies on intercellular adhesion. I. Adhesion of neural retina cells to isotypic aggregates in serum free medium
Authors: Pessac Bernard , Alliot Françoise , Girard Arlette
Published: February 2005

A quantitative assay for intercellular adhesion, which is a modification of the collecting aggregate assay (Roth et al., 1971) is described. The use of mechanically dissociated single cells labeled with 3H‐leucine, and of a gyratory shaker increased considerably the efficiency of cell collection. With chick embryo neural retina, it was shown that isotypic cell adhesion occurs in simple synthetic media and even in sodium chloride solution suggesting that divalent cations do not play a major role in adhesion of these cells. Cell collection in media without serum was not affected by metabolic or protein synthesis inhibitors. However, cell adhesion was temperature‐dependent since no collection occurred at 4°C but was maximal at 38°C.
Electron probe microanalysis of chemical elemental content of single human red cells
Authors: Lechene C. P. , Bronner C. , Kirk R. G.
Published: February 2005

Sodium, potassium, iron and sulfur contents of single human red cells were measured using electron microprobe microanalysis. Three preparative procedures were compared, and the most reliable technique was found to be spraying of cells onto polished pyrolytic graphite by atomization. Primary standards were prepared by adjusting the intracellular electrolyte content of red cells, eliminating the need to correct for X‐ray absorption. Samples were stable under the electron beam during analysis, and could be stored for long periods of time. Strong correlations were found between the X‐ray intensities of iron and sulfur and between potassium and sodium. X‐ray intensities of potassium and sodium were found to be directly proportional to internal ionic content. Large populations of single cells could be analyzed and the distribution of their elemental content studied.
Serum‐stimulated phosphate uptake and initiation of fibroblast proliferation
Authors: Greenberg Deborah B. , Barsh Gregory S. , Ho Tsung‐Shang , Cunningham Dennis D.
Published: February 2005

Previous studies have shown that initiation of proliferation of density‐inhibited fibroblasts by fresh serum is accompanied by a rapid increase in phosphate uptake. This increase might be a key event in the initiation of DNA synthesis. The present studies examined this possibility.

Mouse 3T3, secondary chick embryo, or human diploid foreskin cultures were grown to quiescence in medium containing varying levels of serum. When proliferation of the cultures was initiated by addition of fresh serum, the changes in phosphate uptake were inversely related to the final increases in cell number. Additional experiments showed that the change in phosphate uptake following serum addition was determined by the level of phosphate uptake prior to serum addition.

Addition of dexamethasone to quiescent 3T3 cultures caused them to proliferate but did not increase phosphate uptake. Similarly, trypsin or insulin stimulated proliferation of quiescent secondary chick embryo cultures, but caused little or no change in phosphate uptake.

Quiescent 3T3 cultures switched to medium containing fresh serum and reduced levels of phosphate showed a decrease in both phosphate uptake and intracellular phosphate pool size. Cell proliferation in these cultures, however, was stimulated to the same degree as cultures switched to medium containing fresh serum and the normal amount of phosphate. In addition, quiescent secondary chick embryo cultures switched to medium containing fresh serum and no phosphate showed a decrease in the intracellular phosphate pool size. Thymidine incorporation and final cell number in these cultures, however, was stimulated to the same or higher degree than in cultures switched to medium containing fresh serum and the normal amount of phosphate. These results demonstrate that the rapid increase in phosphate uptake following addition of fresh serum to quiescent fibroblasts is not a necessary event for the initiation of proliferation.
Growth and biochemical characteristics of a detachment variant of CHO cells
Authors: Atherly Alan G. , Barnhart Benjamin J. , Kraemer Paul M.
Published: February 2005

A variant subline of Chinese hamster cells (line CHO) was isolated that had increased resistance to detachment from the substratum. Comparisons between parental and variant cells of the complex carbohydrates liberated during trypsin detachment showed that the variant cells synthesized little or no hyaluronic acid. These cells also had reduced amounts of other complex carbohydrates in the cell periphery. However, parental and variant cells did not differ in morphology, growth control, or cyclic AMP concentration. Profound changes in the physical and chemical nature of the cell periphery, in themselves, evidently are insufficient to cause changes in many aspects of cell behavior.
The relative amounts of the cytoplasmic RNA species in normal, transformed and senescent cultured cell lines
Authors: Johnson Lee F. , Abelson Herbert T. , Penman Sheldon , Green Howard
Published: February 2005

We have examined the relative quantities of 18S and 28S rRNA, 4S RNA and poly (A) + mRNA in the following cultured cells: the mouse fibroblast lines 3T3 and 3T6 in the resting (contact inhibited) and growing (sparse) states, 3T3 clones transformed with SV40 (SV3T3) and with both SV40 and polyoma (SV‐Py 3T3), hamster lung fibroblasts (V79), human cervical carcinoma cells (HeLa), and human diploid fibroblasts at early and late passage. The relative quantities of the RNA species were determined by labeling the cells to equilibrium with 32PO4 and measuring the amount of label in each RNA species.

The ratio of mRNA to rRNA varied from 1.1% to 2.7% in the different cell lines, the more rapidly growing cell lines usually giving a higher ratio. In cells experiencing growth limitation either by contact inhibition or due to senescence, the ratio of mRNA to rRNA was about 30% lower than in the corresponding cells in the growing state. In most cell lines the ratio of 4S RNA to 18S rRNA was between 0.8 and 1.2, but in senescent fibroblasts, this ratio increased to greater than 1.7. Senescent fibroblasts also contained much more total RNA per unit of DNA than the same cells at early passage or than 3T6 or 3T3 cells.
The influence of culture conditions on the induction of tyrosine aminotransferase by cyclic nucleotides in rat hepatoma cells
Authors: Stellwagen Robert H. , Kohli Kulwant K. , Sailor Rochelle D.
Published: February 2005

The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2′‐dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8‐substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.
The binding sites of cytochalasin D. II. Their relationship to hexose transport and to cytochalasin B
Authors: Tannenbaum Janet , Tanenbaum Stuart W. , Godman Gabriel C.
Published: February 2005

Cytochalasin B (CB) was able to compete with tritiated cytochalasin D (3H‐CD) for binding sites in HEp‐2 cells. The pattern of inhibition suggested that CB associates with a low affinity class of CD binding sites. Glucose and maltose did not inhibit binding of 3H‐CD to isolated HEp‐2 plasma membrane. Inhibition of hexose transport by CD was negligible, but CD did not block the potent inhibition of this transport by CB. These results indicate that CD does not bind to the high affinity CB receptor reportedly associated with the hexose transport system, and that this receptor cannot mediate the morphological effects of CD. Both CD and CB induced contraction‐zeoisis in HEp‐2 cells; CB was less potent than CD, and their effects appeared to be additive. It was concluded that the high affinity binding sites for CD and CB are different, but that these congeners share a low affinity site. Both high and low affinity sites for CD appear to mediate its morphological effects; only the low affinity class appears to be involved for CB. Possible identification of the common low affinity binding site as actomyosin (detailed in Tannenbaum et al., 1977) is further discussed.
The diverse effects of 5′‐bromodeoxyuridine on enzyme activities in cultured H35 hepatoma cells
Authors: Gurr James A. , Becker Joyce E. , Potter Van R.
Published: February 2005

Reuber (H35) hepatoma cells were grown in medium containing 10 −5M bromodeoxyuridine (BrdU), which was incorporated into their DNA. Cell growth rate was unaffected by BrdU for the first two generations, after which it was reduced by about 50%. The effect of BrdU incorporation on the activities of several enzymes with rapid turnover rates was examined to test the hypothesis that the synthesis of such enzymes will be preferentially inhibited by BrdU. Tyrosine amino‐transferase (TAT) activity decreased by 70% within two generations whereas thymidine kinase activity remained at control values. PEP carboxykinase activity was unchanged during the first generation in BrdU‐containing medium but, during the second, its activity increased by at least 30%. Ornithine decarboxylase levels decreased by about 50% only after two generations in the presence of BrdU. There appeared to be no simple relationship between turnover rates and the effect of BrdU on enzyme activity. Incorporation of BrdU was found to inhibit the induction of both TAT and PEP carboxykinase by dexamethasone and to enhance the inhibition of cell growth by this steroid. These results are discussed with respect to possible mechanisms of gene expression and development in both normal and neoplastic cells.
Effects of temperature, metabolic and cytoskeletal inhibitors on the rate of BHK cell adhesion to polystyrene
Authors: Nath Kamalendu , Srere Paul A.
Published: February 2005

Adhesion of baby hamster kidney fibroblasts (BHK cells) to a Falcon tissue culture flask was measured under various physiological conditions. While 75–80% of the fibroblasts adhere at temperatures from 19–50°, cellular adhesion decreased dramatically below 19°. Less than 10% of the cells adhere to the substratum even after prolonged incubations at temperatures of 8° or below. This lack of adhesion at low temperatures cannot be overcome by the application of increased gravitational force to the cells.

No correlation exists between cellular ATP concentrations or respiration rates and the rate of cell adhesion to the substratum. One millimolar Na F and 1 mM 2,4 dinitrophenol together lower cellular ATP concentration by 95% but adhesion is reduced by only 50%. NaN3 and KCN greatly lower cellular ATP concentrations without a corresponding inhibition of adhesion. Inhibition of cellular respiration by these compounds occurs at lower concentrations than does the inhibition of adhesion. Two micrograms/milliliters of cytochalasin B inhibits adhesion by 90%, 0.1 mM vinblastine sulphate or colchicine by less than 50% and 50 μg/ml colcemid by less than 30%.

Fixing the cells with formaldehyde, hardening their membranes with ZnCl2 or treating the cells with toluene, all cause an inhibition in adhesion. Again, application of increased gravitational force cannot overcome these latter inhibitions of BHK cell adhesion to the surface of the flasks.
The adhesion of Chinese hamster cells. I. Effects of temperature, metabolic inhibitors and proteolytic dissection of cell surface macromolecules
Authors: Juliano R. L. , Gagalang E.
Published: February 2005

We have investigated factors controlling the rate of adhesion of suspension culture CHO cells to serum coated glass surfaces. Thus, we have examined (1) the effect of metabolic depletion via KCN treatment; (2) effects of colchicine and cytochalasin‐B, two drugs which are presumptive inhibitors of microtubule and microfilament activities, respectively; (3) effects of temperature on adhesion; (4) the correlation between changes in cell adhesion rate and alterations of lacteroperoxidase labelled cell surface proteins consequent to treatment with proteases.

We have found that colchicine is completely without effect on the initial adhesion process in CHO cells, indicating that microtubules are not a determinant of adhesion kinetics. Cytochalasin‐B and depletion of ATP stores by KCN both diminish CHO cell adhesion, but seem to act in different ways. Treatment with cytochalasin‐B results in a dose dependent reduction in the initial slope of adhesion versus time curves, while KCN treatment produces a pronounced lag period during which little cell attachment takes place, followed by a relatively rapid rise to control levels of cell attachment.

Analysis of CHO cell adhesion kinetics as a function of temperature reveals that the inhibition produced by low temperature seems similar to that produced by KCN. Thus at elevated (25°C) temperature, CHO cells adhere rapidly, while at 4°C adhesion is completely inhibited. However, at intermediate temperatures (10°C) a pronounced lag period followed by a rapid rise is noted, a result similar to that seen in KCN treated cells.

Treatment of CHO cells with low doses (10 μg/ml) of crystalline trypsin results in the loss of several major cell surface proteins, without any appreciable effect on adhesion kinetics. High doses (100–1,000 μg/ml) of trypsin result in the cleavage of other proteins whose loss may be related to a parallel loss in adhesive ability. Some of the CHO cell surface proteins seem resistant to doses of trypsin (1,000 μg/ml) which completely abolish the ability of cells to adhere.

Our observations on the effects of inhibitors, temperature, and surface proteolysis on CHO cell adhesion kinetics suggest the involvement of (a) microfilaments, (b) plasma membrane fluidity, and (c) a subclass of the surface proteins as important determinants of the adhesion process.
The inhibition by colchicine of the initiation of DNA synthesis by hepatocytes in regenerating rat liver and by cultivated WI‐38 and C3H10T 1/2 cells
Authors: Roy Walker P. , Boynton Alton L. , Whitfield James F.
Published: February 2005

Colchicine inhibited the initiation of DNA synthesis by hepatocytes in vivo and by WI‐38 and C3H10T 1/2 cells in vitro. All three cell types were most sensitive to colchicine when it was administered at the time of, or shortly after, proliferative activation (by partial hepatectomy or medium‐serum change). WI‐38 and C3H10T 1/2 cells became less sensitive to colchincine as the time between proliferative activation and addition of the drug was increased. Hepatocytes, on the other hand, showed a second stage of sensitivity immediately before the onset of DNA synthesis. Ongoing DNA synthesis was not inhibited by colchicine in any of the cell types. The two prereplicative stages of colchicine sensitivity in liver were also sensitive to vincristine but not to lumicolchicine, an analogue of colchicine which does not bind to tubulin. The data obtained with these drugs indicates that microtubules may be involved in the prereplicative stages of cell proliferation, but non‐specific binding to other membrane proteins cannot be ruled out. Colchicine (at a dose of 50 μg/100 g of body weight), which when injected 30 minutes after hepatectomy delayed the initiation of DNA synthesis for at least 14 hours, delayed the induction of ornithine decarboxylase (at the beginning of prereplicative development) by only two hours. Thus, the inhibitory action of colchicine on the initiation of DNA synthesis does not appear to be mediated by inhibition of polyamine synthesis.
A comparison of the responses of cultured myoblasts and chondrocytes to fibroblast and epidermal growth factors
Authors: Gospodarowicz Denis , Mescher Anthony L.
Published: February 2005

The effects of fibroblast and epidermal growth factors on proliferation and differentiation of cultured myoblasts and chondrocytes have been compared. FGF stimulated myoblast proliferation, as determined by monitoring levels of DNA synthesis during seven days growth in vitro and by the morphology of the cultures after myotube formation. EGF has relatively little effect on myoblast proliferation. With chondrocytes, both FGF and EGF are mitogenic and FGF's, but not EGF's effect is potentiated by dexamethasone. One implication of these results is that in the course of differentiation cell types which develop from the same embryonic origin as fibroblasts are controlled by different sets of mitogenic factors. Myoblasts become primarily dependent on mitogenic agents such as FGF while chondrocytes can respond to both FGF and EGF.
The mechanism of killing of mouse fibroblasts by the amino acid analogue 5‐fluorotryptophan
Authors: Taub Mary
Published: February 2005

The mechanism of killing of A9 fibroblasts by 5‐fluorotryptophan has been studied. L‐tryptophan competitively relieves the growth inhibition caused by 5FT. After incubation with 5FT, 3H‐5FT was incorporated into protein, replacing tryptophan residues. During the initial hours of incubation with 5FT, a specific inhibition was observed of the incorporation of L‐tryptophan into protein; later this inhibition was followed by a general inhibition of protein synthesis and cell division. However, nuclear division continued after cell division had ceased. While 5FT was observed to be incorporated into protein after a 1 hour period in MEM + 0.40 mM 5FT in A9, no 3H‐5FT was incorporated into protein in a mutant isolated by its resistance to killing by 5FT. These results support the hypothesis that cell death occurs due to malfunctioning proteins which contain 5FT residues.