bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibronectin‐associated transforming growth factor
Authors: Fava R. A. , McClure D. B.
Published: February 2005

We have studied the ability of fibronectins to induce anchorage‐independent growth of NRK‐49F cells in serum‐free medium. Cells were seeded in soft agar in the presence of various concentrations of plasma fibronectins, and colonies were counted after 10 days. It was found that, with some exceptions, human plasma fibronectins induced anchorage‐independent growth at concentrations in 20–100 μg/ml range. The ability of exogenously supplied fibronectins to promote anchorage‐independent growth of NRK cells is attributed to a transforming growth factor (TGF) activity associated with gelatin‐agarose affinity purified plasma fibronectins. This TGF activity required epidermal growth factor (EGF) in our serum‐free assay system. The TGF‐like activity appears to either co‐purify or to be associated with fibronectin at neutral pH during molecular sieve chromatography and during ultracentrifugation through sucrose density gradients. The TGF activity “dissociates” from fibronectin at extremes of pH, however, and can be separated from fibronectin by molecular sieve chromatography in 1 M acetic acid. Under these conditions, the TGF‐like activity chromatographed as a single peak with an apparent molecular weight of approximately 30 kDa. The physical‐chemical properties, chromatographic behavior, and biological activity of this TGF suggest that it is type‐β transforming growth factor/growth inhibitor (β‐TGF/GI). The TGF activity has been observed in fibronectin isolated from fresh human plasma as well as in fibronectins from several other species obtained from commercial suppliers. Our results would suggest that caution be applied in the interpretation of experiments in which gelatin affinity purified fibronectins are used at μg/ml concentrations.
Functional differentiation of virgin mouse mammary epithelium in explant culture is dependent upon extracellular proline
Authors: Smith Gilbert H.
Published: February 2005

Depletion of proline from insulin, hydrocortisone, and prolactin‐containing medium prior to incubating virgin mouse mammary explants prevents both DNA synthesis and functional differentiation in the mammary epithelial cells; however, DNA synthesis in the mammary stroma and total incorporation of radioactive amino acids into total protein appears to continue without hindrance. Removal of glycine instead of proline had no deleterious effect on either DNA replication in the hormone‐stimulated epithelium or in its functional differentiation. Functional differentiation was determined by the induction of casein and α‐lactalbumin synthesis in the insulin, hydrocortisone, and prolactin (IFPrl)‐treated explant cultures. As a control, the induction of mouse mammary tumor virus (MMTV) gene expression, a cortiocosteroid‐regulated function, was also measured. Neither the absence of proline or glycine prevented the glucocorticoid stimulation of MMTV gene expression. In contrast to mammary tissue from virgin mice, explants from nonpregnant primiparous mice responded fully to IFPrl stimulation with respect to DNA, casein, and α‐lactalbumin synthesis in medium depleted of proline. These data suggest that the uncommitted epithelium of virgin mouse mammary glands requires the presence of exogenous proline in order to respond to lactogenic hormonal signals. We have demonstrated earlier that DNA synthesis is a prerequisite of functional differentiation in virgin mouse mammary explants (Smith and Vonderhaar, 1981, Dev. Biol., 88:167–179; Vonderhaar and Smith, 1982, J. Cell Sci, 53:97–114), although cytological differentiation proceeded unencumbered in explants prevented from synthesizing DNA. Here, without proline, neither cytological nor functional differentiation can be induced; this suggests that proline provides an essential metabolic interlock in the acquisition of lactogenic hormone responsiveness in uncommitted mouse mammary tissue.
The role of benzodiazepine receptors in the induction of differentiation of HL‐60 leukemia cells by benzodiazepines and purines
Authors: Ishiguro Kimiko , Taft William C. , DeLorenzo Robert J. , Sartorelli Alan C.
Published: February 2005

A series of benzodiazepines was evaluated for their capacity to induce the differentiation of HL‐60 acute promyelocytic leukemia cells. Benzodiazepines were effective initiators of maturation in the concentration range of 50 to 150 μM. The possible involvement of benzodiazepine receptors in mediating the differentiation induced by these agents was investigated. The presence of high affinity, peripheral type benzodiazepine binding sites (KD = 7.3 nM, TB = 14.5 pmol/mg protein with Ro5–4864) was demonstrated in HL‐60 membranes. The occupancy of peripheral type high affinity benzodiazepine receptors by various benzodiazepines showed some correlation (r = 0.76) with their differentiation‐inducing capabilities, but binding potencies were 1,000‐fold higher than the concentrations required to produce differentiation. A class of benzodiazepine receptors with lower binding affinity was also detected in HL‐60 membranes (KD = 28.6 μM; TB = 199 pmol/mg protein with diazepam). A higher level of correlation (r = 0.88) was demonstrated between benzodiazepine occupancy of these lower affinity receptors and the capacity to induce maturation. Significantly, benzodiazepine concentrations needed for low affinity binding and induction of differentiation were the same (25–200 μM), suggesting that low affinity benzodiazepine receptors may be involved in the induction process. We have shown that the molecular form responsible for the induction of the differentiation of HL‐60 cells to mature forms by 6‐thioguanine (TGua) is the free base, TGua, itself [Ishiguro, Schwartz, and Sartorelli (1984) J. Cell. Physiol., 121:383–390]. Since hypoxanthine (Hyp) and inosine (Ino) have been identified as putative endogenous ligands for high affinity benzodiazepine receptors in brain tissue, the potential involvement of benzodiazepine receptors in the differentiation of HL‐60 cells by the purines was investigated. Physiological purines such as Hyp and Ino were inactive in displacing the benzodiazepines from their high and low affinity binding sites in HL‐60 membranes. In contrast, TGua caused inhibition of benzodiazepine binding to high and low affinity sites. The inhibition of Ro5–4864 binding to high affinity binding sites by TGua appeared to be due to the binding of TGua to membranes through the formation of a mixed disulfide between the 6‐thiopurine and protein thiols, since the inhibition was reversed by the presence of 2‐mercaptoethanol. The findings suggest a possible relationship between the occupancy of benzodiazepine receptors by TGua and the induction of leukemic cell differentiation.
γ‐glutamyl transpeptidase (γ‐GT) and maintenance of thiol pools in tumor cells resistant to alkylating agents
Authors: Ahmad Shakeel , Okine Laud , Wood Robert , Aljian John , Vistica David T.
Published: February 2005

Previous studies from this laboratory have established that acquired resistance of murine L1210 leukemia cells to L‐phenylalanine mustard (L‐PAM) and other alkylating agents is accompanied by a two‐to threefold elevation in their glutathione (GSH) concentration (Biochem. Pharm. 31:121). In an attempt to gain insight into the mechanism by which resistant tumor cells maintain their increased GSH content, we have assessed the possible role of γ‐glutamyl transpeptidase (γ‐GT), a membrane bound enzyme involved in GSH metabolism. These results indicate that the enzyme is present in both sensitive and resistant murine L1210 leukemia cells but that the cellular content of γ‐GT is elevated two‐to threefold in L‐PAM resistant cells as compared to their sensitive counterparts. This elevation in enzymatic activity correlates well with the increased cellular GSH content in resistant cells. The results of a detailed kinetic analysis of γ‐GT activity indicate that there is no difference, between cell types, in the apparent Km of the enzyme for the γ‐glutamyl donor (L‐γ‐glutamyl‐p‐nitroanilide) or the acceptor (glycylglycine). However, the apparent Vmax is increased two‐to threefold in L‐PAM resistant tumor cells. Investigation into the role of γ‐GT in the extracellular metabolism of GSH indicates that resistant tumor cells metabolize two‐fold more GSH than do sensitive cells and that such metabolism results in a similar difference in the intracellular concentration of cysteine. Results of studies with cellular lysates also indicate a role for the enzyme in the supply of cysteine to the glutathione precursor pool of the tumor cell and in the maintenance of elevated GSH concentrations in cells resistant to alkylating agents.
Modulation of cellular heat sensitivity by specific amino acids
Authors: Vidair Charles A. , Dewey William C.
Published: February 2005

When either plateau‐phase or exponentially growing Chinese hamster ovary (CHO) cells are incubated in amino acid‐free medium, the cells become sensitized to killing by heat. For cells deprived of amino acids for 12 h survival decreases from 1 × 10−2 for controls to 1 × 10−6 for the deprived cells, following heating at 45°C for 38 min. The survival of these sensitized cells is rapidly increased by the addition of a single amino acid just prior to heating. Of the 21 amino acids which are added in purified form to make McCoy's 5a medium, 12 show no protective effect, four have a small protective effect, and either alanine, asparagine, glutamine, serine, or theronine raise survival to a level similar to that of the control cells. The nonmetabolizable alanine analogue, 2‐aminoisobutyric acid (AIB), increases survival of amino acid‐deprived cells as effectively as each member of the group of five listed above, suggesting that metabolic conversion of the amino acids is not required for their protective effect. The data suggest that an increase in the intracellular concentrations of specific amino acids, independent of any change in cellular ATP content or the rate of protein synthesis, enables these cells to become quickly more resistant to killing by heat. We also conclude that the amino acid concentrations in poorly vascularized regions of some tumors should be considered, along with the oxygen, glucose, and proton concentrations, as factors which determine cellular survival following hyperthermia.
Characterization of the receptor for protease nexin‐I: Protease complexes on human fibroblasts
Authors: Howard Eric W. , Knauer Daniel J.
Published: February 2005

Fibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media. Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets. Protease nexin‐l, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes. Human fibroblasts bind complexes of protease nexin‐l and its target protease via a cell‐surface, high‐affinity receptor. We have analyzed a number of characteristics of this receptor, and found them to be typical of class II receptors in general. At 4°C binding of PN‐l:protease complexes was competable by heparin. In addition, binding was independent of the particular protease bound to the PN‐l; purified complexes of PN‐l with thrombin or urokinase competed equipotently for [125]l‐thrombin:PN‐l binding. As the pH of the binding buffer was lowered, binding to cells increased. A twofold increase in binding was attained by lowering the pH from 7.5 to 4.5. This phenomenon was not due to irreversible, pH‐induced changes to either the cell surface or the labeled complexes. At 37°C, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions. The internalization of complexes was also very rapid, with an estimated ke (endocytic rate cor stant) of 1.0 min−1 At neutral pH, fibroblasts bind complexes in a saturable manner. Scatchard analysis yields areceptor number of 250,000 per cell and a Kd of 1 nM.
Differential control of mRNA levels for Thy‐1 antigen and laminin in rat mammary epithelial and myoepithelial‐like cells
Authors: Barraclough Roger , Kimbell Rosemary , Rudland Philip S.
Published: February 2005

Thy‐1 antigen and laminin are two components often associated with the basement membrane of the rat mammary gland and are thought to be synthesized, at least in part, by the adjacent myoepithelial cells in vivo. The relative levels of Thy‐1 mRNA and laminin mRNA are compared in a rat mammary cuboidal epithelial cell line and a derivative elongated myoepithelial‐like cell line by hybridizing cloned cDNAs to cellular mRNA isolated from these cell types. Although the elongated myoepithelial‐like cells synthesize four times as much laminin protein as the cuboidal epithelial cells, there is only a 1.7‐fold increase in laminin mRNA between the two cell types. In contrast the 17‐fold increase in Thy‐1 antigen between the elongated cells and the cuboidal cells can be accounted for completely by a 14–18‐fold increase in Thy‐1 mRNA, suggesting that changes in the steady‐state levels of Thy‐1 mRNA in these cell lines are modulated at either a transcriptional or a post‐transcriptional level. Run‐off transcription by nuclei isolated from the cell lines does not distinguish between these two possibilities. The comparative results on Thy‐1 antigen and laminin show that the enhanced production of two proteins often associated with the basement membrane of the rat mammary gland can be controlled at different levels in the elongated myoepithelial‐like cells.
Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell associated and platelet‐derived growth factor‐like protein which is secreted
Authors: Vlodavsky Israel , Fridman Rafael , Sullivan Robert , Sasse Joachim , Klagsbrun Michael
Published: February 2005

Cultured bovine aortic endothelial cells synthesize growth factors which markedly differ in the regulation of their storage and secretion. Endothelial cell lysates, but not conditioned medium, contain a growth factor activity that appears to be basic fibroblast growth factor (FGF) by the following criteria: (1) it elutes from heparin‐Sepharose at 1.4–1.6 M NaCl; (2) it is mitogenic for bovine aortic and capillary endothelial cells; (3) it is heat sensitive but stable to dithiothreitol; (4) it has a molecular weight of about 18,000 daltons; and (5) it cross‐reacts with antiserum directed against basic FGF. In contrast, endothelial cell conditioned medium, but not lysates, contains a growth factor activity that (1) elutes from heparin‐Sepharose at 0.4 – 0.5 M NaCl; (2) is mitogenic for fibroblasts and vascular smooth muscle cells but not for capillary endothelial cells; (3) is heat stable and dithiothreitol sensitive; and (4) competes with platelet‐derived growth factor (PDGF) for binding to fibroblasts. From these criteria, it appears that endothelial cells secrete into the medium growth factors some of which are PDGF‐like, but secrete little if any basic FGF. It is suggested that endothelial cell‐associated basic FGF acts in an autocrine fashion to stimulate endothelial cell proliferation in response to endothelial cell perturbation or injury. On the other hand, the endothelial cell‐secreted growth factors which are smooth muscle cell but not endothelial cell mitogens might exert a paracrine function on neighboring cells of the vessel wall.
Effect of phagocytosis on receptor distribution and endocytic activity in macrophages
Authors: Buys Saundra S. , Kaplan Jerry
Published: February 2005

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or lgG‐coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, α2‐macroglobulin · protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage‐like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor‐mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor‐mediated ligand uptake.
Modulation of murine erythroleukemic cell differentiation by inhibitors of polyamine biosynthesis
Authors: Meilhoc Eliane , Moutin Marie‐José , Osborne H. Beverley
Published: February 2005

The differentiation of murine erythroleukemic cells induced by hexamethylene bisacetamide is shown to be differently affected by two inhibitors of polyamine biosynthesis. Methyl glyoxal bis(guanyl hydrazone) (inhibitor or S‐adenosyl methionine decarboxylase) inhibited this differentiation process. By using a novel experiment protocol the inhibitory effect of this drug on the induced differentiation was dissociated from pleiotropic effects on cell growth. Methyl glyoxal bis(guanyl hydrazone) only inhibited the induced differentiation if present during the first 6 h of culture of the cells with the inducer. No effect on the induced differentiation was observed if the drug was added to the culture medium 6 h after the inducer. α‐Difluoro methylornithine (inhibitor of ornithine decarboxylase) stimulated the differentiation of these cells. Polyamine analysis demonstrated that α‐difluoro methylornithine increased the rapidity and the amplitude of the changes in intracellular polyamines associated with this induced differentiation. The presence of methyl glyoxal bis(guanyl hydrazone) during the first 3 h with the inducer was sufficient to produce opposing changes in the intracellular polyamines. These results suggest that changes in either intracellular polyamines or the activities of polyamine biosynthetic enzymes play a regulatory role in the differentiation process induced in murine erythroleukemic cells by hexamethylene bisacetamide.
Biological activity, binding, and metabolic fate of Ac‐[Nle4, D‐Phe7]α‐MSH4–11NH2 with the F1 variant of B16 melanoma cells
Authors: Panasci L. C. , McQuillan A. , Kaufman M.
Published: February 2005

The α‐MSH (α‐melanocyte‐stimulating hormone) agonist, Ac‐[Nle4, D‐Phe7]α‐MSH4–11NH2 (hereafter called ND4–11α‐MSH), is at least 10‐fold more potent than α‐MSH as a stimulus of tyrosinase activity in F1 variant cells of B16 melanoma. The binding to these cells during an incubation with 5 nM (3H)ND4–11α‐MSH at 37°C is maximal at 0–30 min, 22 fmol/106 cells, but declines to 40% of this value at 4 hr. In the presence of 5 nM (3H)ND4–11α‐MSH at 37°C, the acid soluble (cell surface) radioactivity decreased rapidly from 11.4 fmol/106 cells at 5 min to 4.6 fmol/106 cells at 4 hr. Chromatographic analysis of media and cellular samples revealed that there was no evidence of degradation of (3H)ND4–11α‐MSH in the medium but there was evidence of intracellular degradation of (3H)ND4–11α‐MSH. Ammonium chloride (10mM) resulted in an increase in acid resistant radioactivity (internalized hormone) at 4 hr.

The binding to F1 variant cells during an incubation with 0.155 nM or 5 nM (3H)ND4–11α‐MSH at 4°C was constant from 4 hr to 24 hr. Under these conditions, there was no time‐dependent change in the acid soluble radioactivity from 4 to 24 hr. Scatchard analysis of (3H)ND4–11α‐MSH binding to F1 variant cells at 4°C demonstrated that there were approximately 4500 receptors per cetl and an association constant of 17.1 nM−l. These results are consistent with a process of (3H)ND4–1α‐MSH binding to its receptor followed by internalization of the receptor‐hormone complex and then intracellular degradation of the hormone.
Suramin inhibition of growth factor receptor binding and mitogenicity in AKR‐2B cells
Authors: Coffey Robert J. , Leof Edward B. , Shipley Gary D. , Moses Harold L.
Published: February 2005

Suramin, a polyanionic compound, has previously been shown to dissociate platelet‐derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR‐2B cells in fetal bovine serum (FBS)‐supplemented medium in a dose‐dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor β (TGFβ), heparin‐binding growth factor type‐2 (HBGF‐2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density‐arrested cultures of AKR‐2B cells. The inhibition of growth factor‐stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I‐labeled TGFβ, HBGF‐2, and EGF to their cell surface receptors. Suramin affected TGFβ and HBGF‐2‐related events at a 10–15‐fold lower dose than that required for EGF‐related events. It was also noted that suramin inhibited TGFβ‐stimulated soft agar colony formation of AKR‐2B (clone 84A) cells as well as the spontaneous colony formation of AKR‐MCA cells, a chemically transformed derivative of AKR‐2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGFβ, HBGF‐2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR‐MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.
Bicarbonate/chloride antiport in vero cells: II. Mechanisms for bicarbonate‐dependent regulation of intracellular pH
Authors: Olsnes Sjur , Ludt Jannikke , Tønnessen Tor Inge , Sandvig Kirsten
Published: February 2005

The rates of bicarbonate‐dependent uptake and efflux of 22Na+ in Vero cells were studied and compared with the uptake and efflux of 36Cl−. Both processes were strongly inhibited by DIDS. Whereas the transport of chloride increased approximately ten‐fold when the internal pH was increased over a narrow range around neutrality, the uptake of Na+ was much less affected by changes in pH. The bicarbonate‐linked uptake of 22Na+ was dependent on internal Cl− but not on internal Na+. At a constant external concentration of HCO−3, the amount of 22Na+ associated with the cells increased when the internal concentration of HCO−3 decreased and vice versa, which is compatible with the possibility that the ion pair NaCO−3 is the transported species and that the transport is symmetric across the membrane. Bicarbonate inhibited the uptake of 36Cl− both in the absence and presence of Na+. At alkaline internal pH, HCO−3 stimulated the efflux of 36Cl− from preloaded cells, while at acidic internal pH both Na+ and HCO−3 were required to induce 36Cl− efflux. We propose a model for how bicarbonate‐dependent regulation of the internal pH may occur. This model implies the existence of two bicarbonate transport mechanisms that, under physiological conditions, transport OH−‐ equivalents in opposite directions across the plasma membrane.
Fibronectin stimulates growth but not follicle‐stimulating hormone‐dependent differentiation of rat granulosa cells in vitro
Authors: Morley Paul , Armstrong David T. , Gore‐Langton Robert E.
Published: February 2005

Since fibronectin is a secretory product of immature rat granulosa cells in culture and may contribute to the follicular microenvironment in vivo, we have studied the effects of this adhesion factor on follicle‐stimulating hormone (FSH)‐dependent differentiation in short‐term (2–3‐day) cultures and on growth and protein synthesis in long‐term (12‐day) cultures. In comparison with cells plated on tissue culture plastic, those plated on an optimal fibronectin‐coated substratum showed much greater cell spreading. There were no short‐term effects of this morphological change on FSH‐stimulation of cyclic AMP production, apparent activities of aromatase or cholesterol side‐chain cleavage enzymes, or acquisition of luteinizing hormone (LH) responsiveness in cultured cells. However, progesterone metabolism to 20α‐hydroxypregnan‐4‐en‐3‐one was increased. Only cultures on fibronectin showed increases between days 3 and 9 in protein (2.5‐fold) and DNA (1.4‐fold) contents. Cells cultured on fibronectin also showed greater uptake and incorporation of [3H]leucine in comparison with cells cultured on plastic. FSH treatment caused cell aggregation and rounding and delayed the increase in protein content of cells cultured on fibronectin. The results presented demonstrate that the principal direct effect of fibronectin‐mediated adhesion on rat granulosa cells is to enhance cell maintenance and growth, while having no generalized action on FSH‐dependent differentiation.
Control of the adipogenic differentiation of 3T3‐F442A cells by retinoic acid, dexamethasone, and insulin: A topographic analysis
Authors: Pairault Jacques , Lasnier FrançOise
Published: February 2005

Differentiation of 3T3‐F442A adipocytes, monitored by accumulation of neutral lipid and by using the sensitive marker glycerophosphate dehydrogenase, is inhibited by incubation of confluent 3T3‐F442A fibroblasts in medium containing retinoic acid or dexamethasone. When added together, dexamethasone (0.25 μM) potentiates about 50‐fold the inhibitory effect of retinoic acid (10 μM). Insulin cannot counteract the retinoic acid blockade; however, it can overcome the inhibition of differentiation elicited by dexamethasone. These differential effects of insulin are used for characterizing the adipose conversion cycle. We describe cell culture conditions where terminal differentiation of 3T3‐F442A preadipocytes is achieved by low, physiological levels of insulin. They include the switch from a high‐serum medium containing isobutyl methyl xanthine and dexamethasone to a serum‐free, hormone‐supplemented medium. The data reported establish the existence of two successive states for commitment to adipogenic differentiation: a first commitment point (CA) to differentiation which requires serum adipogenic factors, and a second commitment point (CH) controlled by lipogenic hormones, namely insulin, after which terminal maturation can resume. We demonstrate that retinoic acid can prevent and interrupt differentiation by blocking the cells within the early differentiation phase.
Protamine sulfate inhibits mitogenic activities of the extracellular matrix and fibroblast growth factor, but potentiates that of epidermal growth factor
Authors: Neufeld G. , Gospodarowicz D.
Published: February 2005

Protamine sulfate, an inhibitor of angiogenesis in vivo, markedly inhibits the ability of angiogenic factors such as acidic or basic fibroblast growth factor (aFGF, bFGF) to stimulate the proliferation in vitro of either BHK‐21 cells or vascular endothelial cells. The inhibition is reversible, and cells remain viable even after prolonged exposure to protamine sulfate. Protamine sulfate inhibits the mitogenic effects of both growth factors by preventing them from binding to their common cell surface receptors. It also inhibits the mitogenic activity of the extracellular matrix produced by bovine corneal endothelial cells. This substrate has been shown in previous studies to replace the requirement for FGF of many cell types. In contrast, protamine sulfate potentiates the mitogenic activity of epidermal growth factor (EGF). This indicates that protamine sulfate also acts at cellular sites which are not associated with FGF receptors.
The effect of factors released from the tumor‐transformed cells on DNA synthesis, mitosis, and cellular enlargement in 3T3 fibroblasts
Authors: Dafgård Eva , Engström Wilhelm , Larsson Olle , Zetterberg Anders
Published: February 2005

Quiescent serum‐starved 3T3 cells can be stimulated to initiate DNA synthesis after addition of conditioned media from spontaneously tumor‐transformed 3T3 cells (3T6‐cells) or from SV‐40‐transformed 3T3 cells (SV‐3T3 cells). The conditioned media were found to stimulate both the chromosome cycle (i.e, DNA synthesis and cell division) and the growth cycle (i.e., cellular enlargement). Furthermore, addition of conditioned media to quiescent 3T3 cells increased the activity of HMG CoA reductase—an enzyme previously proposed to exercise some control on cell proliferation in 3T3 cells Larsson and Zetterberg: J. Cell. Physiol. 129:99–102, 1986. The increased activity of HMG CoA reductase after treatment with tumor cell conditioned media was correlated to the stimulatory effects on DNA synthesis. By treating 3T3 cells stimulated to resume proliferation by addition of conditioned media with mevinolin (a competitive inhibitor of HMG CoA reductase) the activity of HMG CoA reductase as well as the DNA synthesis and cell division were efficiently inhibited. In contrast, HMG CoA activity was not coupled to the cellular enlargement. Therefore, it is proposed that one set of factors present in tumor cell conditioned media preferentially stimulates the chromosome cycle by increasing the HMG‐CoA reductase activity, whereas another set of factors is responsible for growth in cell size. Both types of factors are required for balanced growth.
The effects of sulfhydryl inhibitors and cytochalasin on the cytoplasmic and cytoskeletal actin of human neutrophils
Authors: Wallace Paul J. , Packman Charles H. , Wersto Robert P. , Lichtman Marshall A.
Published: February 2005

To better understand the changes that occur in cytoplasmic actin during cell movement, we studied the effect of inhibitors of cell movement on the molecular conformation of actin and its incorporation into the Triton‐insoluble cytoskeleton of human neutrophils. The sulfhydryl reactive compound N‐ethylmaleimide caused an increase in cellular F‐actin as measured by uptake of the F‐actin specific fluorescent probe 7‐nitrobenz‐2‐oxadiazole‐phallacidin. However, N‐ethylmaleimide reduced the amount of actin associated with the Triton‐insoluble cytoskeleton. Dithiobisnitrobenzoic acid, a sulfhydryl reagent that does not cross cell membranes efficiently, did not alter the F‐actin content of neutrophils. The effect of N‐ethylmaleimide was blocked by the presence of dithiothreitol, a donor of sulfhydryl groups. N‐ethylmaleimide did not affect the polymerization of actin in a cell‐free system. Cytochalasin B did not alter F‐actin content of neutrophils but did decrease actin in cytoskeletons of resting neutrophils. Cytochalasin inhibited the increase in F‐actin initiated by the chemoattractant N‐formylmethionylleucylphenylalanine. We propose that N‐ethylmaleimide blocks the stabilization of G‐actin in cytoplasm, interferes with the incorporation of F‐actin polymer into the cytoskeleton, and depolymerizes the cytoskeleton. In contrast cytochalasin stabilizes G‐actin in the presence of chemotactic peptide. These data suggest that reversible conversion of G‐actin to F‐actin and incorporation of F‐actin into the Triton‐insoluble cytoskeleton are important for neutrophil movement.
Cell‐cycle‐dependent expression of human ornithine decarboxylase
Authors: Kaczmarek Leszek , Calabretta Bruno , Ferrari Sergio , de Riel Jon K.
Published: February 2005

A human ornithine decarboxylase (ODC) gene probe has been isolated from a Jurkat T‐cell cDNA expression library, sequenced, and used to analyze ODC mRNA levels in untransformed human lymphocytes and fibroblasts stimulated to proliferate by various mitogens. The partial cDNA sequence is 86% homologous to the mouse ODC cDNA, and Northern blots indicate that the human and mouse mRNA species are similar in size. ODC mRNA is barely detectable in quiescent human T lymphocytes and undetectable in density‐arrested WI38 fibroblasts. Following stimulation of T‐lymphocyte proliferation with phytohemagglutinin, the ODC mRNA level rises to a peak around mid G1 phase and decreases as the cells enter S phase. Serum stimulation of density‐arrested fibroblasts results in an elevation of the ODC mRNA level which persists throughout the cell cycle. Epidermal growth factor (20 ng/ml) but not insulin (10 mg/ml) or dexamethasone (55 ng/ml) stimulates ODC expression in quiescent WI38 fibroblasts. Southern blots suggest that human cells have a single copy of the ODC gene.
Purified monocyte‐derived angiogenic substance (angiotropin) stimulates migration, phenotypic changes, and “tube formation” but not proliferation of capillary endothelial cells in vitro
Authors: Höckel Michael , Sasse Joachim , Wissler Josef H.
Published: February 2005

Activated monocytes (macrophages, histiocytes) induce the formation of new blood vessels by secretion product(s). From conditioned serum‐free media of porcine peripheral monocytes treated with concanavalin A, a substance with very strong angiogenic activity in vivo, designated as angiotropin, has been isolated and purified to homogeneity. We investigated the biological action of the monocyte‐derived angiogenic substance on cultured capillary and large vessel (aorta) endothelial cells and on 3T3 fibroblasts, mimicking steps of the angiogenic pathway in vitro. We found that angiotropin does not stimulate the proliferation of capillary endothelial and 3T3 cells; however, in concentrations less than 1 ng/ml, it enhances random migration of capillary endothelial cells but not of 3T3 cells. On confluent monolayers of capillary and aortic endothelial cells angiotropin leads to defined changes of cell morphology that are dose dependent and reversible. In the presence of angiotropin, capillary endothelial cells rapidly form tubelike structures on gelatinized plates. This organizational state is not reached with aortic endothelial cells. The results indicate that the biological action of monocytic angiotropin is different from that of the angiogenic growth factors that stimulate the proliferation of endothelial cells and nonlymphoid mesenchymal cells and keep endothelial cells in the contact‐inhibited epitheloid cell phenotype. We propose that angiotropin is directly involved in monocyte‐induced angiogenesis.
Increased adherence of oxidant‐treated human and bovine erythrocytes to cultured endothelial cells
Authors: Wali Ramesh K. , Jaffe Stuart , Kumar Dinesh , Sorgente Nino , Kalra Vijay K.
Published: February 2005

Bovine erythrocytes, which normally lack phosphatidyl choline in their membranes, when treated with either H2O2 or diamide (1–3 mM), showed a partial appearance of phosphatidyl ethanolamine (PE 40%) and phosphatidyl serine (PS, 30–33%) in the external leaflet of the bilayer and a concomitant increased (four‐ to five‐fold) propensity to adhere to cultured bovine aortic endothelial cells. Similar treatment of normal human erythrocytes caused an alteration in the organization of the phospholipid bilayer and also resulted in their increased adherence to endothelial cells derived either from human umbilical vein or bovine aorta. Treatment of RBCs with H2O2 at low concentration (0.5 mM) resulted in cross‐linking of spectrin without significant changes in the orientation of aminophospholipids but the RBCs exhibited 15–20% increase in adherence to endothelial cells. Pretreatment of either human or bovine erythrocytes with antioxidants such as vitamin E (2 mM) prevented both oxidant‐induced reorganization of phospholipids in the bilayer and enhancement of adherence to endothelial cells. Introduction of either phosphatidyl serine or phosphatidyl ethanolamine but not phosphatidyl choline into erythrocyte membranes increased their adherence to endothelial cells threefold. Oxidant‐treated RBCs exhibited enhanced binding and fluorescence of Merocyanine 540 dye (MC‐540), which is sensitive to the packing of lipids in the lipid bilayer. On flow cytometric analysis, 78% of H2O2 (0.5 mM)‐treated erythrocytes compared to 30% of untreated RBCs exhibited MC‐540 binding and fluorescence, indicating differences in the lipid packing in the outer leaflet of the bilayer. Oxidant‐treated erythrocytes adhere preferentially to endothelial cells rather than to bovine aortic smooth muscle cells and skin fibroblasts. It is suggested that the alterations in the erythrocyte membrane surface due to spectrin cross‐linking and the organization of the phospholipids concomitant with less ordered packing in the external leaflet of the bilayer, either induced by oxidative manipulation in normal RBC or in pathological erythrocytes, play a role in erythrocyte‐endothelial cell interaction.
Isolation of a clone of F9 teratocarcinoma cells “naturally” resistant to G418
Authors: Edwards S. A. , Adamson E. D.
Published: February 2005

Resistance to the neomycin analogue G418 forms the basis of a dominant marker selection system for mammalian (and other) cells transfected with the bacterial neo gene. This system has been particularly effective because of the low incidence of spontaneous conversion to G418 resistance in mammalian cells; no case of resistance to the drug in the absence of the bacterial genes has yet been reported to our knowledge. During the course of transfection experiments, we recently isolated a clone of F9 teratocarcinoma cells which is drug resistant yet has no detectable integrated plasmid sequences, neo RNA transcripts, or aminoglycoside phosphotransferase activity. The G418‐resistant clone (F9nr7) did not display enhanced resistance to other cytotoxic drugs tested: colchicine, actinomycin D, cycloheximide, and hygromycin B. Therefore, nr7 cells differ from multidrug‐resistant phenotypes previously described. However, this clone is inhibited, relative to control cells, in its response to the differentiation‐inducing drugs retinoic acid and dibutyryl cAMP, which suggests that some aspects of general drug metabolism may be altered in these cells.
Ultraviolet radiation directly induces pigment production by cultured human melanocytes
Authors: Friedmann Peter S. , Gilchrest Barbara A.
Published: February 2005

In humans the major stimulus for cutaneous pigmentation is ultraviolet radiation (UVR). Little is known about the mechanism underlying this response, in part because of the complexity of interactions in whole epidermis. Using a recently developed culture system, human melanocytes were exposed daily to a physiologic range of UVR doses from a solar simulator. Responses were determined 24 hours after the last exposure. There was a dose‐related increase in melanin content per cell and uptake of 14C‐DOPA, accompanied by growth inhibition. Cells from donors of different racial origin gave proportionately similar increases in melanin, although there were approximately tenfold differences in basal values. Light and electron microscopy revealed UVR‐stimulated increases in dendricity as well as melanosome number and degree of melanization, analogous to the well‐recognized melanocyte changes following sun exposure of intact skin. Similar responses were seen with Cloudman S91 melanoma cells, although this murine cell line required lower UVR dosages and fewer exposures for maximal stimulation. These data establish that UVR is capable of directly stimulating melanogenesis. Because cyclic AMP elevation has been associated in some settings with increased pigment production by cultured melanocytes, preliminary experiments were conducted to see if the effects of UVR were mediated by cAMP. Both α‐MSH and isobutylmethylxanthine (IBMX), as positive controls, caused a fourfold increase in cAMP level in human melanocytes and/or S91 cells, but following a dose of UVR sufficient to stimulate pigment production there was no change in cAMP level up to 4 hours after exposure. Thus it appears that the UVR‐induced melanogenesis is mediated by cAMP‐independent mechanisms.
Mutant KB cells with decreased EGF receptor expression: Biochemical characterization
Authors: Hwang Jaulang , Richert Nancy , Pastan Ira , Gottesman Michael M.
Published: February 2005

Mutants of the human KB carcinoma cell line resistant to a cytotoxic conjugate of epidermal growth factor and Pseudomonas exotoxin (EGF‐PE) express a pleiotropic phenotype, which includes reduced levels of 125I‐EGF binding, without altered affinity for EGF (Lyall et al., 1987). Here, the EGF‐toxin (ET) resistant mutants were further characterized with respect to the amount and size of the EGF receptor and the level of EGF receptor RNA. These data indicate that decreased binding of 125I‐EGF in the mutants is due to reduced amounts of EGF receptor, which is associated with decreased mRNA levels. Changes in other proteins in the ET mutants were also examined. Five of the six ET mutants had a decrease in a 78,000 Mr− membrane glycoprotein. In addition, an increase in a protein with a Mr− of 40,000 and a pl = 8.0 was found in all the mutants, and an increase in a series of proteins with a Mr− of 36,000 and a pl of 6.3–6.5 was found in some of the mutants. These results confirm the pleiotropic nature of the EGF‐PE resistant mutants and show that reduced EGF binding is due to altered expression of the EGF receptor gene in the mutants.
Apurinic site induction in the DNA of cells heated at hyperthermic temperatures
Authors: Warters Raymond L. , Brizgys Lucy M.
Published: February 2005

The induction of DNA damage in cells heated at hyperthermic (43–48°C) temperatures was determined by alkaline filter elution and alkaline sucrose gradient‐sedimentation analysis of cell DNA denatured at pH 13.0. A class of DNA lesion which converted to strand breaks during denaturation of DNA at pH 13.0 was produced randomly throughout the cell DNA at temperatures as low as 43°C. Induction of this lesion occurred with a T0 of 90 and 10 min at 45 and 48°C, respectively. We estimate that these pH 13.0–detectable DNA lesions are produced in the cell DNA with a frequency of approximately 75 and 660 per min of heating at 45 and 48°C, respectively. Since the lesions were quantitatively converted to DNA strand breaks at pH 13.0 with a half‐time of 30 min, or less, we suggest that these pH 13.0–detectable DNA lesions are heat‐induced, abasic DNA sites. The induction of these lesions does not appear to be directly involved in the initial heat‐induced inhibition of DNA synthesis. The presence of these lesions cannot be excluded as an explanation for the long‐term inhibition of replicon initiated in heated cells.
Human breast epithelial cells in serum‐free collagen gel primary culture: Growth, morphological, and immunocytochemical analysis
Authors: Yang Jason , Balakrishnan Arun , Hamamoto Susan , Elias Joel J. , Rosenau Werner , Beattie Craig W. , Das Gupta Tapas K. , Wellings Sefton R. , Nandi Satyabrata
Published: February 2005

Human breast epithelial cells derived from various sources (fibroadenoma, reduction mammoplasty, and mastectomy tissues from premenopausal patients) have been cultured in collagen gel matrix using serum‐free medium. Response to various additives has been analyzed for growth‐promoting effect when added to a basal medium containing insulin, cholera toxin, and BSA. A consistent observation has been the effect of EGF and cortisol in growth stimulation of human breast epithelial cells, while separately, each additive elicited only a small response. Under this condition, employing EGF and cortisol combinations, these cells gave rise to organized colonies consisting of clusters of cells, usually spherical, without any duct‐like extensions. Ultrastructural and immunocytochemical studies, using a panel of monoclonal and polyclonal antibodies, have shown that cell types and features that can be identified in the original breast tissue can also be delineated in the progeny populations. The topographical feature, consisting of lumina surrounded by a single inner layer of epithelial cells and an outer layer of basal/myoepithelial cells, can be re‐created in the collagen gel system starting from small clumps of cells.
Maintenance of periportal and pericentral oxygen tensions in primary rat hepatocyte cultures: Influence on cellular DNA and protein content monitored by flow cytometry
Authors: Holzer Claudia , Maier Peter
Published: February 2005

Primary hepatocytes were cultured at oxygen tensions similar to those reported to be present in periportal (13% O2) and pericentral (4% O2) regions of the liver lobules. Cellular DNA and protein content of individual hepatocytes were determined simultaneously by two‐parameter (DNA/protein) flow cytometry after 1, 4, and 7 days in culture. PO2 tensions monitored on line in conventional plastic culture dishes revealed that the depletion of the PO2 in the culture medium depended on the number of hepatocytes plated. When cultured as monolayer after 4–7 days at periportal (13% O2) and more pronounced at pericentral oxygen concentration (4% O2), up to 90% of the hepatocytes showed degenerated nuclei but normal protein content. By using culture dishes with teflon membrane bottoms the oxygen tension in the culture medium was accurately maintained by the incubator atmosphere. At pericentral oxygen tension the fraction of 2N cells increased by about 20%. That of the 4N cell was not affected, and the contribution of 8N hepatocytes dropped to 70% compared to cultures at periportal oxygen tension. Concomitantly, in the 4% O2 hepatocyte cultures the protein content of the 2N and the 4N cells was better preserved and increased by up to 10%. These results suggest that in vitro at pericentral oxygen conditions (4% O2) ageing of hepatocytes is delayed, regenerating processes are better maintained, and, furthermore, freshly isolated 4N hepatocytes have the potency to adapt their metabolism in vitro to periportal as well as to perivenous oxygen tensions.
Ornithine decarboxylase activity during rat spermatogenesis in vivo and in vitro: Selective effect of hormones and growth factors
Authors: Smith Frank F. , Tres Laura L. , Kierszenbaum Abraham L.
Published: February 2005

We have established the patterns of ornithine decarboxylase activity (an enzyme related to cell growth, differentiation, and proliferation) during rat testicular development and studied the effect of epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), transforming growth factor‐type β (TGF‐β), and a serum‐free, hormone/growth factor‐supplemented medium (TKM) on ornithine decarboxylase (ODC) activity in Sertoli‐spermatogenic cell cocultures and cultured seminiferous peritubular cells prepared from sexually immature rats (20–22 days old). Results were correlated with timing of ODC activities during rat testicular development. We have found that: (1) although EGF, alone or combined with PDGF and TGF‐β, and TKM stimulated ODC activity in Sertoli‐spermatogenic cell cocultures after 6 and 24 h of stimulation, PDGF exerted an inhibitory effect, and (2) cultured peritubular cells stimulated with EGF, PDGF, TGF‐β (and their combinations), and TKM displayed an increase in ODC activity after 6 h of stimulation, but ODC activities for most of these treatments declined considerably 24 h after stimulation. Light microscopic autoradiographic studies of [3H]thymidine labeled samples demonstrated that (1) clones of spermatogenic cells traverse S phase synchronously, (2) Sertoli cells are not significantly radiolabeled, probably because of contact inhibition achieved by high cell plating density, and (3) peritubular cells are significantly [3H]thymidine labeled in the presence of TKM, a culture medium that facilitates spermatogenic cell long‐term viability and differentiation. We conclude that TKM and EGF have stimulatory effects on the biochemical pathway that precedes synchronous DNA synthesis in spermatogonia and preleptotene spermatocytes, and that ODC activity is a sensitive marker for monitoring these events.