bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitogenic and antimitogenic effects of cholera toxin‐mediated cyclic AMP levels in 3T3 cells
Authors: Smets Lou A. , Van Rooy Henny
Published: February 2005

The effect of time‐controlled exposures to cholera toxin (CT) on intracellular levels of cyclic AMP (cAMP) and on the proliferative response of serum‐stimulated 3T3 cells was investigated. Continuous exposure to CT caused up to 8‐fold raises in cAMP content and inhibited DNA replication by delaying G1‐S transition and by reducing the fraction of cells committed to DNA replication. In contrast, short exposures to CT during G0‐G1 transition increased the fraction of cells responding to serum stimulation and potentiated the serum‐induced morphological changes in the cell monolayer. A short exposure during late G1 phase, however, inhibited the onset of DNA synthesis but had little effect on ongoing DNA replication. The results indicate that cAMP has diverse and opposite effects on two defined restriction points in cell cycle control. Cyclic AMP was positively involved in the acquistion of the state of competence by quiescent cells (G0‐G1 transition) but antagonistic on the onset of DNA replication (G1‐S transition) in committed cells. The observations reconcile a number of controversial conclusions regarding the role of cAMP in cell cycle control.
Type β transforming growth factor (TGFβ) regulation of alkaline phosphatase expression and other phenotype‐related mRNAs in osteoblastic rat osteosarcoma cells
Authors: Noda Masaki , Rodan Gideon A.
Published: February 2005

TGFβ1 from porcine platelets increased alkaline phosphatase (AP) activity in the rat osteoblastic cell line ROS 17/2.8 about three‐fold. This effect was dose‐dependent with an ED50 of about ∼0.2 ng/ml and was larger during logarithmic growth than at confluence. TGFβ1 inhibited cell growth by about 30% with similar dose dependence. Thirty min exposure to TGFβ1 was sufficient to increase AP activity 3 days later by about two‐fold but did not affect cell growth, suggesting dissociation between effects on proliferation and differentiation. The rise in AP activity started 6 h after TGFβ1 addition and was blocked by cycloheximide and actinomycin D. TGFβ1 also increased AP mRNA by two‐ to three‐fold and this effect was not blocked by cycloheximide. The half‐life of AP mRNA, estimated following the addition of 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole was about ten h in both control and TGFβ1‐treated cells. The mRNAs for type I procollagen and osteonectin were also increased by TGFβ1 but fibronectin mRNA was decreased. TGFβ2 effects on AP and cell growth were similar to those of TGFβ1, except for lack of activity following transient exposure. At saturating concentrations, TGFβ2 (2 ng/ml) or dexamethasone (10−7M), which has similar effects on these cells, did not further augment the effects of TGFβ1 (at 2 ng/ml). Above findings suggest that TGFβ promotes osteoblatic differentiation in rat osteosarcoma cells at least in part by acting at the pretranslational level.
Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor‐beta
Authors: Allen Ronald E. , Boxhorn Linda K.
Published: February 2005

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with transforming growth factor‐beta (TGF‐beta). Muscle‐specific protein synthesis and satellite cell fusion were used as indicators of muscle differentiation; a dose‐dependent inhibition of differentiation was observed in response to TGF‐beta. In addition, TGF‐beta depressed cell proliferation in a dose‐dependent manner. Half‐maximal inhibition of differentiation was seen with a TGF‐beta concentration of approximately 0.1 ng/ml. Although proliferation was not inhibited, it was depressed and half‐maximal suppression of proliferation occurred in response to 0.1–0.5 ng TGF‐beta/ml. Neonatal rat myoblasts were also subjected to TGF‐beta treatment, and similar results were observed. Neonatal cells, however, were more sensitive to TGF‐beta than satellite cells, as indicated by the reduced concentrations of TGF‐beta required to inhibit differentiation and reduce the rate of proliferation. Under identical culture conditions proliferation of muscle‐derived fibroblasts were also depressed. The differentiation inhibiting effect of TGF‐beta on satellite cells was reversible. It has been suggested that TGF‐beta could be an important regulator of tissue repair, and its in vitro effects on satellite cells suggest a possible role in regulation of muscle regeneration.
Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I
Authors: Harvey Gillian , Pearson Colin K.
Published: February 2005

Confirmatory evidence for the existence of a multienzyme complex of DNA precursor pathways in mammalian cells was obtained. Using neutral sucrose gradient centrifugation of cell lysates we found that at least five enzymes involved in DNA precursor metabolism in uninfected, S‐phase BHK‐cell fibroblasts cosediment at a common rate, indicative of a multienzyme complex. The enzymes include DNA polymerase thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and NDP‐kinase. This complex was partially, but not completely, disrupted when lysates from G0‐phase cells were centrifuged. Using lysates from cells infected with herpes simplex virus (HSV) type I some of the virus‐induced ribonucleotide reductase and a minor proportion of the HSV‐thymidine kinase cosedimented rapidly. The virus‐induced DNA polymerase sedimented independently near the middle of the gradient, in contrast to the behaviour of the host polymerase. The enzyme associations observed were disrupted by NaCl or by inclusion of ethylenediamine tetraacetic acid during the cell lysis procedure, instead of the usual EGTA. These results indicate the importance of ionic forces in maintaining the enzyme complexes. The bulk of the DNA and the RNA present in the lysates did not sediment at the same rate as the complexes, showing that the enzymes were not simply adhering nonspecifically to these polyanions. Newly synthesised radiolabeled DNA (15 min pulse with [3H]thymidine) was not preferentially associated with the enzymes, but some functional DNA was evident in the enzyme complex fraction from the uninfected S‐phase cells. DNA polymerase activity in this fraction did not require, nor was it stimulated by, exogenous “activated” DNA. Added DNA primer‐template was required, however, for maximal activity of the polymerase in gradient fractions derived from G0‐phase cells and from HSV‐infected cells. No evidence for channeling of ribonucleotide precursors into DNA of permeabilized cells (uninfected or HSV‐infected) was detected. Most rCDP was incorporated into RNA. In the uninfected, S‐phase cells about 10 pmol/106 cells/90 min of rCDP residues was incorporated into DNA compared with 120 pmol/106 cells/90 min when radiolabeled dCTP was used. Nonradioactive dCTP present in equimolar concentration in the incubation with labeled rCDP did not, however, diminish the incorporation of label from the ribonucleotide. In permeabilized HSV‐infected cells incorporation of radiolabel from rCDP into DNA was barely detectable.
Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: Comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase α activity
Authors: Otto Angela M. , Smith Christina , De Asua Luis Jimenez
Published: February 2005

An approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2α insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca+ +‐containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine‐triphosphate for 1 hr at 37°C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)‐precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density‐labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole‐cell lysates prepared with a hypotonic buffer not containing Ca+ +. Using various templates, it was shown that the increase in activity of DNA polymerase α correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase β activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.
Comparison of tyrosinase levels in amelanotic and melanotic melanoma cell cultures by a competitive enzyme‐linked immunoadsorbent assay and by immunotitration analysis
Authors: Fuller Bryan B. , Iman Deborah S. , Lunsford Julie B.
Published: February 2005

Melanogenesis in mammalian pigment cells is regulated by changes in the activity of tryosinase, the rate‐limiting enzyme for melanin synthesis. Because recent evidence suggests that this enzyme may exist in pigment cells in both active and inactive stages, a competitive enzyme‐linked immunoadsorbent assay (ELISA) was developed to compare tyrosinase levels in amelanotic and melanotic melanoma cell clones. The melanotic cell line used for this study, MEL‐11A, had basal tyrosinase levels approximately 40 times that of the amelanotic cell line, AM‐7. Both cell lines responded to melanocyte‐stimulating hormone by demonstrating large increases in tyrosinase activity. For competitive ELISA analysis of tyrosinase levels in these two clones, microtiter plates were coated with purified tyrosinase, and trypsinized cell extracts were tested for their ability to compete with bound tyrosinase for antibody binding. Although tyrosinase activity in the amelanotic clone was 1/40 that of the melanotic clone, immunoreactive tyrosinase levels in AM‐7 cells were found to be approximately one‐half that present in the melanotic clone. Additional evidence for the presence of an inactive (or at least, catalytically less active) enzyme in AM‐7 cells was obtained from immunotitration analysis of tyrosinase in cell extracts from both cell lines. These results suggest that at least some amelanotic melanoma cells may contain significant levels of catalytically inactive tyrosinase molecules and that the level of pigmentation in mammalian melanocytes may be regulated by a tyrosinase activation process.
Stimulation of mono (ADP‐ribosyl)ation by reduced extracellular calcium levels in human fibroblasts
Authors: Duncan Michael R. , Rankin Patrick R. , King Robert L. , Jacobson Myron K. , Dell'Orco Robert T.
Published: February 2005

Lowering extracellular calcium in cultures of human diploid fibroblast‐like cells caused a rapid depletion of NAD pools. This loss of NAD was reversed by restoring extracellular Ca2+ and was inhibited by 3‐aminobenzamide, an inhibitor of ADP‐ribosyl transfer reactions. The concentrations of 3‐aminobenzamide needed to inhibit the loss of NAD were consistent with those required to inhibit cellular mono(ADP‐ribosyl) rather than poly(ADP‐ribosyl) reactions. Calcium depletion did not inhibit the biosynthesis of NAD. These results suggest that mono(ADP‐ribosyl)ation is involved in the regulation of cellular Ca2+ levels.
Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells
Authors: Schleef Raymond R. , Wagner Nancy V. , Loskutoff David J.
Published: February 2005

This report describes the development and use of functional immunoradiometric assays that distinguish the activity of β‐migrating endothelial‐type plasminogen activator inhibitor (PAI‐1) from that of placental‐type plasminogen activator inhibitor (PAI‐2). These assays are based upon the binding of PAI‐1 and PAI‐2 to immobilized single‐chain tissue‐type plasminogen activator (tPA) and to immobilized urokinase (UK), respectively. The extent of binding of each PAI is quantified by incubating the PAI‐PA complex first with rabbit antiserum specific for the individual PAI and then with 125I‐labeled goat antirabbit IgG. In control experiments, the assays were shown to be sensitive, dose‐dependent over a wide range, and specific for each PAI. These assays were employed to establish the PAI profile of a variety of human cells. Neither PAI‐1 nor PAI‐2 could be detected in Bowes melanoma cells or in a renal adenocarcinoma cell line (ACHN), while the histiocytic lymphoma cell (U‐937) produced only PAI‐2. Five cell lines, including two that were previously shown to contain one or the other PAI (e.g., umbilical vein endothelial cells and a fibrosarcoma cell line, HT‐1080) in fact contained both PAIs. The cells containing both PAIs were studied in more detail. In each case, SDS treatment of CM was shown to enhance PAI‐1 activity (by converting the latent form of this inhibitor into its active form) and to destroy PAI‐2 activity. Various compounds including interleukin 1, dexamethasone, and phorbol myristate acetate were found to selectively influence the cellular production of one PAI without concomitantly affecting the production of the other, suggesting that the synthesis of these inhibitors is not coordinately regulated.
A phorbol ester‐nonproliferative variant of Swiss 3T3 cells is deficient in Na+ K+ Cl− cotransport activity
Authors: O'Brien Thomas G. , Prettyman Ralph , George Kenneth S. , Herschman Harvey R.
Published: February 2005

The identity of the genetic defect(s) in Swiss 3T3 TNR‐2 and TNR‐9 that confers nonresponsiveness to the proliferative effect of 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) is not known. In BALB/c 3T3 cells, loss (via mutation) of a specific membrane ion transport system, the furosemide‐sensitive Na+ K+ Cl− cotransporter, is associated with decreased responsiveness to TPA. In this study, the transport properties of parental Swiss 3T3 cells and the TPA‐nonresponsive lines TNR‐2 and TNR‐9 were determined in the presence and absence of TPA. When the rate of 86Rb+ efflux (as a tracer for K+) was measured from each of the three cell lines, a furosemide‐ and TPA‐inhibitable component of efflux was clearly evident in parental and TNR‐9 cells but was virtually absent in TNR‐2 cells. 86Rb+ influx measurements indicated the presence in parental 3T3 cells and the TNR‐9 line of a substantial furosemide‐sensitive flux that could be inhibited by TPA. In contrast, much less furosemide‐sensitive influx was present in 3T3‐TNR‐2 cells, and it was relatively unaffected by TPA. In both parental 3T3 and 3T3‐TNR‐2 cells, most of the furosemide‐sensitive 86Rb+ influx is dependent on extracellular Na+ and Cl−. The apparent affinities of the transporter for these two ions, as well as for K+, were similar in both cell lines. In parental cells, the inhibition of furosemide‐sensitive 86Rb+ influx was quite sensitive to TPA (K1/2 ≅ 1 nM) and occurred very rapidly after phorbol ester exposure. As expected because of its volume‐regulatory role, inhibition of Na+ K+ Cl− cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of cotransport activity in TNR‐2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3‐TNR‐2 cells (but not TNR‐9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+ K+ Cl− cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of contransport activity in TNR‐2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3‐TNR‐2 cells (but not TNR‐9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+ K+ Cl− contransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.
Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix
Authors: Schuetz Erin G. , Li Donna , Omiecinski Curtis J. , Muller‐Eberhard Ursula , Kleinman Hynda K. , Elswick Barbara , Guzelian Philip S.
Published: February 2005

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver‐specific functions over time in culture. We incubated hepatocytes in a serum‐free medium on a reconstituted basement membrane gel, “matrigel” (prepared from an extract of extracellular matrix of the murine Engelbreth‐Holm‐Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver‐specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, contained higher amounts of glutathione peroxidase, and, as judged by Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver‐specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P‐450b, and cytochrome P‐450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P‐450c by 3‐methylcholanthrene and for P‐450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P‐450d by 3‐methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P‐450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte “dedifferentiation.” None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H‐thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of mRNAs for the cytoskeleton proteins actin and tubulin in hepatocytes on both matrices during the first 2 days in culture. However, the continuously flattening hepatocytes on Vitrogen maintained substantially higher levels of cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver‐specific functions.
Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases
Authors: Montesano R. , Pepper M. S. , Belin D. , Vassalli J.‐D. , Orci L.
Published: February 2005

We have previously shown that capillary endothelial cells grown on the surface of three‐dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary‐like tubular structures in response to tumor‐promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine‐specific phosphatases, to determine whether this agent could induce the expression of an anglogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasrninogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase‐type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.
Expression of the CSF‐1 gene in the blast cells of acute myeloblastic leukemia: Association with reduced growth capacity
Authors: Wang Chen , Kelleher C. A. , Cheng G. Y. M. , Miyauchi Jun , Wong Gordon G. , Clark Steven C. , Minden Mark D. , McCulloch Ernest A.
Published: February 2005

Myelopoietic growth factors are known to influence the growth in culture of malignant blast cells from human Acute Myeloblastic Leukemia (AML). We have used cDNA clones for the factor CSF‐1 and its receptor fms to study DNA and RNA from the blasts of 25 AML patients. The CSF‐1 gene was always in the germline configuration. CSF‐1 mRNA was found in about half the blast populations. The cells were also studied for their growth properties in culture. A highly significant association was found between CSF‐1 expression and poor growth in suspension culture. Most blast populations expressed fms; the number of fms expression negative samples was too small to permit the detection of any association between fms expression and growth or any interaction between the effects of the expression of the growth factor and its receptor. We propose that CSF‐1 may be an important part of the mechanism determining the balance between self‐renewal and determination in AML blast clones.
End products of glucose and glutamine metabolism by cultured cell lines
Authors: Lanks Karl W. , Li Ping‐Wu
Published: February 2005

Rates of CO2 production from glucose and glutamine, intracellular metabolite levels, and release of metabolic end products into the culture medium were determined for 13 cultured cell lines, including a glycolysis‐defective mutant. All the non‐mutant lines synthesized pyruvate, lactate, alanine, proline, aspartate, and citrate, so that the metabolism of glucose and glutamine resulted mainly in the production of these compounds and only to a lesser extent in complete oxidation to CO2. These data and the pattern of metabolites produced by the mutant line were consistent with a model characterized by incomplete glutamine oxidation leading to end product accumulation. Multiple linear regression analysis identified the metabolite levels most highly correlated with the intracellular citrate level and with the amount of citrate released into the medium. The analysis also showed that the rates of CO2 production from glucose and glutamine were themselves positively correlated, suggesting that the oxidation of the two substrates is coordinately controlled under normal culture conditions.
Transforming growth factor beta (TGF‐β) inhibits hepatocyte DNA synthesis independently of EGF binding and egf receptor autophosphorylation
Authors: Russell William E.
Published: February 2005

Subpicomolar concentrations of human platelet‐derived transforming growth factor beta (TGF‐β) inhibited growth factor‐stimulated DNA synthesis in primary cultures of adult rat hepatocytes. This inhibition was not the result of changes in the size of intracellular pools of 3H‐thymidine and was not dependent on the state of confluence of the cells. A 24‐hr exposure to TGF‐β either before or after insulin/EGF stimulation was as inhibitory on DNA synthesis between 48 and 72 hr of culture as was TGF‐β present throughout 72 hr of culture. From 12 hr in culture to 24 hr, hepatocyte EGF binding sites dropped from about 230,000 to 85,000 per cell with no significant change in Kd, but with a loss in capacity for EGF‐induced receptor down‐regulation. Maximally inhibitory concentrations of TGF‐β did not compete with EGF for the EGF receptor, and a 4‐to 24‐hr exposure to TGF‐β did not alter subsequent EGF binding. Coincubation of hepatocytes with TGF‐β and EGF did not influence the 60% reduction in EGF binding sites produced by EGF alone. In addition, TGF‐β did not prevent EGF‐induced autophosphorylation of the 170,000 dalton EGF receptor in membranes form whole liver. Our studies suggest that TGF‐β regulates hepatocyte growth independently of changes in EGF receptor number, ligand affinity, or postbinding autophosphorylation.
Inositol is a required nutrient for keratinocyte growth
Authors: Gordon Philip R. , Mawhinney Thomas P. , Gilchrest Barbara A.
Published: February 2005

Bovine hypothalamus is known to contain a growth‐promoting activity for human epidermal keratinocytes. By sequential purification, the substance was isolated and found to be myo‐inositol. The identity of the substance as myo‐inositol was confirmed by ion modified partition, gas liquid, thin layer chromatography, by mass spectrometry, and quantitative bioassay. The inositol content of the crude hypothalamic extract and of an active acetone precipitate (the first step in the purification) was determined to be sufficient to account for their observed bioactivity. At an optimal concentration of 55 μM (10 μg/ml), myo‐inositol approximately tripled keratinocyte yield compared to paired cultures in basal medium containing 0.3 μM, although this yield was only half that produced by a crude saline extract of hypothalamus, suggesting that there are additional growth‐promoting activities in the tissue extract. No other skin‐derived cell type tested was stimulated by supplemental inositol. These results establish that the inositol requirement for cultured human keratinocytes is markedly higher than for any other normal or malignant cell type investigated to date, and expand the list of brain‐derived phospholipid precursors known to stimulate epithelial proliferation in vitro. These data suggest that inositol may subserve quantitatively or qualitatively different functions in the keratinocyte than in other cell types.
Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments
Authors: Park Ro Dong , Sullivan Peter C. , Storrie Brian
Published: February 2005

Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes senstive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: (1) in vitro endosomes were osmotically insensitive; and (2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1–10 min at 37°C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate‐conjugated dextran (FITC‐dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC‐dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10‐min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose (“very early” endosomes) and the other not (“early” endosomes). After a brief uptake (1–3 min), postincubation of CHO cells in 0.25 M sucrose‐containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions.
Regulation of the proliferation of primary rat hepatocytes by eicosanoids
Authors: Skouteris George G. , Ord Margery G. , Stocken Lloyd A.
Published: February 2005

DNA synthesis in primary adult rat hepatocyte cultures was promoted by epidermal growth factor (EGF), arachidonic acid, and prostaglandins E2 and F2α (PGE2 and PGF2α). Growth promotion by EGF was blocked by 0.1 mM indomethacin and 1 mM aspirin, without affecting cell viability. If verapamil was present in the medium when EGF was added, the growth response was inhibited. Hepatocytes stimulated by EGF or arachidonic acid released PGE2 and PGF2α into the culture medium. This was diminished if 0.1 mM indomethacin was also in the medium. The importance of autocrine regulation of hepatocyte growth by prostaglandins is discussed.
Heparin‐like molecules regulate the number of epidermal growth factor receptors on vascular smooth muscle cells
Authors: Reilly Christopher F. , Fritze Linda M. S. , Rosenberg Robert D.
Published: February 2005

The effect of heparin on the binding of epidermal growth factor (EGF) to vascular smooth muscle cells (SMC) was examined. Heparin pretreatment of SMC obtained from bovine aortic explant tissue resulted in significant reductions in the amount of EGF bound. Decreases in mitogen binding were observed with both growth arrested as well as exponentially growing cultures. The heparin concentrations (10–100 μg/ml) and pretreatment times (48–72 h) necessary for suppression of EGF binding correlated with the concentrations and temporal requirements necessary for growth inhibition. Chondroitin sulfate, which has negligible antiproliferative activity, had no effect on EGF binding. However, a highly inhibitory heparan sulfate species obtained from postconfluent SMC suppressed EGF binding by 45%. Platelet‐derived growth factor and insulin‐like growth factor‐1 binding were unaffected by heparin. Scatchard analysis revealed that heparin induced 50 to 60% reductions in the numbers of high and low affinity EGF receptors without detectable changes in the binding affinity or ratio of high to low receptors. Experiments were also performed with enzymatically dispersed SMC. These cultures were inhibited by heparin in a time dependent manner which was partially reversible in the presence of EGF. Subsequent studies revealed that heparin suppressed EGF binding in these cultures by 20 to 40%. In summary, heparin reduces the number of EGF receptors on both explant and enzyme dispersed SMC by a mechanism which closely parallels the antiproliferative effects of this glycosaminoglycan.
Stimulation by tumor necrosis factor of HL‐60 thymidine salvage pathway metabolism dissociated from proliferation
Authors: Elias Laurence
Published: February 2005

The effect of human tumor necrosis factor (TNF) on early‐passage HL‐60 cells was studied. A transient phase of increased [3H]thymidine (TdR) incorporation was noted at 20‐24 hr of exposure to TNF. This increase was disproportionate to the much slighter stimulation of the percentage of S‐phase cells, which was measured by flow cytometry. Evidence for increased metabolic trapping of [3H] TdR following TNF treatment was apparent from whole cell uptake experiments. The salvage pathway enzyme TdR kinase was therefore measured and was found to be elevated comparably to [3H]TdR uptake. The mechanism of TNF regulation of TdR kinase was further investigated by a series of combination treatment experiments using other biologic factors and pharmacologic inhibitors of various intracellular steps. The response to TNF was not potentiated or reproduced by IL‐1, IL‐2, IL‐3, IL‐4, G‐CSF, M‐CSF, GM‐CSF or α‐ or γ‐interferon. Blockers of early signal transduction steps, including H7, W7, sphingosine, and pertussis toxin, failed to inhibit TNF stimulation of [3H]TdR incorporation. mRNA synthesis inhibition with α‐amanitin blocked this TNF effect, as did cAMP but not cGMP analogues. A sensitizing effect was noted with amiloride or cytochalasin B, characterized by greater relative increases of [3H]TdR incorporation and TdR kinase activity in response to TNF. In the presence of cytochalasin B, TNF treatment resulted in no change or slight decreases in the percentage of S‐phase cells. Regulation of TdR kinase could thereby be dissociated from the usual cell cycle control. This study thus documents a unique example of stimulation of thymidine salvage pathway metabolism by a biologic factor, dissociable from overall cell cycle regulation.
The effects of in vitro age and culture state on histone variant synthesis in human diploid fibroblasts
Authors: Dell'Orco R. T. , Worthington M. I.
Published: February 2005

Histone variant synthesis patterns from human diploid fibroblast‐like cells of different in vitro ages were determined during exponential growth, at confluence, and during low serum arrest. The results are reported as the ratios of H2A variant synthesis (H2A.1 and H2A.2/H2A.x and H2A.z) and H3 variant synthesis (H3.1 and H3.2/H3.3) that have been used to characterize individual cell cycle states. Hydroxyurea was employed in some experiments to reduce S phase cells. The results indicate that high population doubling level (PDL) cells move through the G1 phase of the division cycle during exponential growth and exist in the G0 cell cycle state at confluence and during low serum arrest. Low PDL cells, however, exist in the G1 cell cycle state at confluence and revert to a G0 state only after maintenance as quiescent populations. This would suggest that when stimulated high PDL cells cannot enter into S phase, they revert to a GO cell cycle state.
Regulation of neuroblast proliferation by hormones and growth factors in chemically defined medium
Authors: Wu Doris K. , Maciag Thomas , de Vellis J.
Published: February 2005

Pure neuronal cultures prepared from 6‐day‐old embryonic chick brains incorporated [3H]‐thymidine in serum‐free medium up to the 4th day in culture. The addition of insulin any time within this culture period caused an increase in thymidine incorporation. This increase in [3H]‐thymidine was correlated with an increase in cell number and percentage of labeling index. Triiodothyronine and endothelial cell growth factor were also active in stimulating [3H]‐thymidine incorporation into chick neuroblasts. The effect of these trophic agents is unique since a variety of known mitogens tested were negative.
Differential expression of cell surface glycoproteins on various organ‐derived microvascular endothelia and endothelial cell cultures
Authors: Belloni Paula N. , Nicolson Garth L.
Published: February 2005

Glycoproteins expressed on the luminal surfaces of microvascular endothelium derived from various murine organs were analyzed and compared with those expressed by cultured vascular endothelial cells. Cell‐surface vascular proteins were radiolabeled in situ via intracardiac perfusion with lactoperoxidase/Na125l. Autoradiography confirmed that the radiolabel was restricted to the vessel lumen in most tissues. Controls contained 125l‐labeled serum proteins to identify adsorbed serum components. Glycoproteins were analyzed by western enzymelinked lectin analysis using detergent extracts of 125l‐labeled microvessels isolated from different organs. The western transfers were probed with a panel of lectin‐peroxidase conjugates to determine differences in protein glycosylation. The same transfers were also screened for exposed 125l‐labeled cell‐surface proteins by autoradiography. This dual analysis detected glycoprotein patterns unique for each organ. At least seven major proteins (Mr ∼ 180 K, 130 K, 95 K, 80 K, 75 K, 60 K, 12 K) were common to microvessels derived from each organ; however, certain glycoproteins appeared to be expressed differentially in particular organs. For example, a Mr ∼ 135 K WGA‐binding glycoprotein was detected in brain microvessels, whereas another WGA‐binding glycoprotein of Mr ∼ 40 K was detected only in kidney. In lung microvessels, a Mr ∼ 140 K WGA binding glycoprotein and a Mr ∼ 55 K RCA‐l‐binding galactoprotein were expressed preferentially, and liver microvessels displayed Mr ∼ 220 K protein and a Mr ∼ 135 K PNA‐binding galactoprotein. The cell‐surface‐iodinated protein profiles from in situ labeled microvessels were similar to profiles derived from cultured bovine aortic endothelial cells and several short‐term endothelial cell cultures isolated from different organs. The results from this study suggest that organ‐associated endothelia express glycoprotein fingerprints unique to each organ.
Influence of the deprivation of a single amino acid on cellular proliferation and survival in rat 3Y1 fibroblasts and their derivatives transformed by a wide variety of agents
Authors: Tanaka Hiroaki , Zaitsu Hirokazu , Onodera Kazukiyo , Kimura Genki
Published: February 2005

We compared proliferation and survival of various syngeneic transformed cell lines under conditions of depletion of 15 amino acids in Dulbecco‐Eagle's medium. We used a normal fibroblast line 3Y1 and 22 transformed sublines of 3Y1 which had been induced by one of seven transforming agents‐simian virus 40, mouse polyomavirus, adenovirus type 12, E1A gene of adenovirus type 12, cDNA of Harvey murine sarcoma virus, Rous sarcoma virus, or N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. Unlike other untransformed cells examined (mouse BALB/c‐3T3 line, mouse NlH‐3T3 line, and primary Fischer rat embryo fibroblasts), 3Y1 ceased to proliferate and accumulated in a viable state with a G1‐phase DNA content under 14 singular deprivations of amino acid. None of the transformed 3Y1 lines completely arrested in the G1 phase of the cell cycle and each showed different levels of survival, depending on each transforming agent. As for transformed 3Y1 cells induced by a given virus or a given transforming gene, any one of the three sublines shared the same trend with respect to proliferation and survival. Transformed derivatives induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine showed almost the same trend in proliferation, but the patterns of survival were not uniform. Our observations suggest that the unique responses of 3Y1 to amino acid depletion are differently modified by different transforming agents.
Normal nonmetastatic human trophoblast cells share in vitro invasive properties of malignant cells
Authors: Yagel Simcha , Parhar Ranjit S. , Jeffrey John J. , Lala Peeyush K.
Published: February 2005

First‐trimester normal human trophoblast cells show some phenotypic similarities to malignant cells, e.g., rapid proliferation and ability to invade neighboring tissue, including basement membrane in situ, but do not have the ability for unlimited growth or metastasis. The present study examined whether the invasive ability of normal trophoblast cells is an intrinsic property of these cells, independent of the microenvironment provided by the pregnant uterus, and if so, whether they share some of the molecular mechanisms of invasion exercized by metastatic malignant cells. The ability of in vitro grown human trophoblast lines to invade an epithelium‐free human amniotic membrane was measured from the temporal kinetics of retention of radioactivity within this membrane resulting from a penetration by 125I‐iododeoxyuridine‐labeled trophoblast cells. The magnitude of this invasion was compared to that of the highly metastatic human JAR‐choriocarcinoma cell line and murine B16F10 melanoma line. Trophoblasts were found to share some of the same molecular mechanisms of invasion with the metastatic cell lines. Inhibitors of collagenase, plasmin, plasminogen, and plasminogen activators completely prevented invasion of the amnion by the trophoblast lines as well as by the metastatic JAR and B16F10 lines. Mersalyl, a compound known to activate collagenase, stimulated invasion by all cell lines tested, including under conditions in which plasmin activity was inhibited. In addition, trophoblasts produced significant levels of type IV collagenase and laminin, both of which appear to be important products of metastatic tumor cells required for basement membrane invasion. It may be concluded from these findings that the invasive property of first trimester human trophoblasts is genetically determined; that the magnitude of amnion invasion cannot differentiate between metastatic cell lines and invasive but nonmetastatic cell lines; and that invasiveness is not a sufficient prerequisite for metastatic ability.
Cultured primate aortic smooth muscle cells express both the PDGF‐‐A and PDGF‐B genes but do not secrete mitogenic activity or dimeric platelet‐derived growth factor protein
Authors: Valente Anthony J. , Delgado Ruby , Metter John D. , Cho Chong , Sprague Eugene A. , Schwartz Colin J. , Graves Dana T.
Published: February 2005

Proliferation of smooth muscle cells (SMC) in the arterial intima of man and experimental animals is important in the pathogenesis of atherosclerosis. Vascular SMC proliferation in vitro is stimulated by a number of agents, including the potent protein mitogen, platelet‐derived growth factor (PDGF). Recent studies on rat arterial SMC indicate that these cells may, under certain circumstances, synthesize PDGF protein mitogens, suggesting that the regulation of SMC proliferation in vivo may have an autocrine or paracrine component. In this study we demonstrate that cultured nonhuman primate (baboon) aortic SMC transcribe both the PDGF‐A and PDGF‐B genes but do not secrete detectable mitogenic activity characteristic of native PDGF. The absence of this activity was not due to the presence in the cell conditioned medium of factors inhibitory for PDGF‐mediated mitogenic activity. Metabolic labeling of the cells and immunoprecipitation with specific antibodies to human PDGF did not detect a dimeric (30 kDa) PDGF protein in either the intracellular or extracellular compartments, but instead identified PDGF‐related proteins of molecular weight 12 kDa and 100 kDa. These data suggest the presence in vascular SMC of a mechanism regulating the translation of PDGF mRNA that may play an important role in the control of SMC proliferation in vivo.
Differential response of normal human fibroblasts to bombesin versus thrombin
Authors: Hendey Bill , Mamrack Mark D.
Published: February 2005

Normal human diploid fibroblasts (WS‐1 cells) were growth‐arrested under serum‐free conditions for 48 hr. The addition of fetal bovine serum (10% final concentration) to these cells stimulated [3H]‐thymidine incorporation into DNA and phosphoinositide breakdown over nine‐fold. Thrombin, at concentrations above 0.1 unit/ml (u/ml), was also effective at stimulating DNA synthesis and phosphoinositide breakdown as well as causing a rise in intracellular pH. In contrast, the peptide bombesin (concentrations ranging from 1 nM to 100 nM) stimulated phosphoinositide breakdown but did not enhance DNA synthesis or cause an increase in cytoplasmic pH. The time course of accumulation of inositol phosphates differed in response to these agents. The thrombin effect peaked rapidly and leveled off after 5 min while the bombesin effect showed a constant increase for 30 min. Serum showed an intermediate response. The different rates of inositol phosphate accumulation observed with the two growth factors is viewed as representing a difference in the mechanism of phosphoinositide turnover. The relationship between the difference in phosphoinositide turnover and the initiation of DNA synthesis is also discussed.
Expression of melanocyte stimulating hormone receptors correlates with mammalian pigmentation, and can be modulated by interferons
Authors: Kameyama Koichiro , Montague Paul M. , Hearing Vincent J.
Published: February 2005

The relationship between melanogenesis and the expression of melanocyte stimulating hormone (MSH) receptors on the surface of melanocytes was examined using sublines generated from the melanotic JB/MS melanoma. JB/MS cells were propagated in long term culture to allow for phenotypic drift in their characteristics of differentiation, and then were cloned; the cloned cells ranged from well differentiated and pigmented to undifferentiated and amelanotic. Spontaneous and MSH‐induced melanogenesis in these different lines was measured and correlated with the number of MSH receptors expressed. After 6 months of in vitro culture, the ability of the cells to respond to MSH was significantly reduced, as were the number of MSH receptors expressed; the cells had reduced pigmentation and were relatively undifferentiated histologically. Subsequently, clonally‐derived pigmented cells were found to have numbers of surface MSH receptors (approximately 60,000 per cell) and levels of melanogenic activity similar to the original JB/MS cell line.

However, an amelanotic clone had an even more dramatically reduced level of pigmentation which correlated with a further decrease in the expression of MSH receptors (< 1,000 per cell) and the production of a potent melanogenic inhibitor. We also examined the responses of these various sublines to α, β, and γ‐interferons and found significant heterogeneity in their abilities to respond to these cytokines. This study clearly shows that there is a direct correlation between melanogenesis and the expression of MSH receptors on the surface of melanocytes, and that melanogenic inhibitors may be critically involved in the regulation of mammalian pigmentation.
Growth factor production by a human megakaryocytic tumor cell line
Authors: Witte David P. , Stambrook Peter J. , Feliciano Estrella , Jones Cindy L. A. , Lieberman Michael A.
Published: February 2005

A recently described human megakaryocytic tumor cell line was analyzed for the presence of growth factor activity and was found to produce large quantities of transforming growth factor b̃‐like (TGF‐b̃) and basic fibroblast growth factor‐like (bFGF) activities. Growth factor activities were identified using a radioreceptor assay for the TGF‐b̃‐like activity, a heparin‐binding assay for the b‐FGF‐like activity, and a demonstration of distinct biological activities for each type of factor. Tumor poly‐A+ RNA revealed strong signals when probed with complementary DNA corresponding to bovine basic FGF and human growth factor (EGF) and TGF‐α. The levels of EGF and TGF‐α produced in the tumor line were too low to be detected by radioreceptor assays. Relative levels of messenger RNA encoding each of the growth factors reflected the relative levels of each of the respective factors tested. These data represent the first definitive identification of FGF‐like activities in megakaryocytic‐like cell lines. Interestingly, the line displayed little activity similar to platelet‐derived growth factor (PDGF) when assayed either biochemically or by poly A+ RNA analysis.