A method for separation of heparin species from biological samples by ethanol precipitation of compounds solubilized in guanidine hydrochloride.

In this paper we describe a procedure to determine glycosaminoglycan and oligosaccharide composition of biological samples such as cell cultures or tissue explants. We demonstrate that heparin species of different molecular mass can be easily fractionated by sequential ethanol precipitation in 4.0 M guanidine hydrochloride. We studied by gradient polyacrylamide gel electrophoresis fractionation of standard heparin and heparin-derived oligosaccharides by anion-exchange chromatography on DEAE-Sephacel resin eluted by increasing concentration of guanidine hydrochloride. The use of guanidine salts followed by sequential precipitation by increasing ethanol concentration allowed recovery of heparin and heparin-derived oligosaccharides.

Heparin binding peptides co-purify with glycosaminoglycans from human plasma.

Glycosaminoglycans (GAGs) are complexed with plasma proteins and proteolysis of plasma reduced the protein-GAG ratio about 140-fold. After dialysis, analysis by gradient PAGE revealed heparinase-1-sensitive GAGs, thus suggesting that heparin could be among the plasma GAGs. However, after dialysis most of the plasma GAGs were still not 'free'. PAGE of peptides resistant to proteolysis showed high molecular weight bands on the two sides of the dialysis membrane despite the 3.5 kDa molecular weight cut-off. Progressive dilution of the sample allowed passage of peptides appearing as high molecular weight bands in the diffusate. We interpret this phenomenon as the presence of low molecular weight peptides that aggregate when concentrated. Peptides on both sides of the membranes bound heparin.

Urokinase-dependent angiogenesis in vitro and diacylglycerol production are blocked by antisense oligonucleotides against the urokinase receptor.

The plasminogen activator system is known to play a crucial role in the angiogenesis process by modulating the adhesive properties of endothelial cells to the extracellular matrix and cell-cell interaction. In the present study, we demonstrated that the urokinase-type plasminogen activator (u-PA) induced neovascular growth in the avascular rabbit cornea and dose-dependently promoted growth, chemotaxis, and matrix invasion of cultured endothelial cells. Interaction between u-PA and its receptor appears to be mandatory for the angiogenic effect of u-PA because monoclonal antibodies anti-u-PA and anti-u-PA receptor (u-PAR) blocked the proangiogenic effects of u-PA at the endothelial cell level. We then assessed the signaling pathway activated in endothelial cells by u-PA. u-PAR activation by u-PA produced de novo synthesis of diacylglycerol (DAG) from glucose by a cytochalasin B-inhibitable mechanism, indicating the involvement of a specific glucose transporter (GLUT). Endothelial cells expressed GLUT2, whose activation was tyrosine kinase-dependent and protein kinase C (PKC)-independent. The increase of glucose uptake led to DAG production, which resulted in PKC activation/translocation. Impairment of u-PAR availability by monoclonal antibodies and by antisense oligonucleotides (aODN) against u-PAR mRNA inhibited glucose uptake, DAG neosynthesis, and PKC activation, resulting in the blockade of endothelial cell proliferation, chemotaxis, and chemoinvasion. These data suggest that u-PAR activation consequent to the binding of u-PA can be regarded as an "angiogenic switch" and disclose the possibility that an anti-u-PAR aODN strategy may efficiently target endothelial cell function to control angiogenesis in vivo.

Premature induction of aging in sublethally H2O2-treated young MRC5 fibroblasts correlates with increased glutathione peroxidase levels and resistance to DNA breakage.

Human MRC5 fibroblasts, at different passages in cultures, were used as an in vitro model to assess variations and/or induction of aging parameters under basal conditions or following sublethal oxidative stress by H2O2. DNA sensitivities to oxidatively-induced breakage, rather than basal levels of damaged DNA, were significantly different between cultures at low and high population doubling level (PDL): old cells maintained most of their DNA integrity even at high concentrations of H2O2, while young cells showed more extensive DNA damage which developed in a dose-dependent fashion. However, young cells pretreated with low doses of H2O2 exhibited increased resistance against further oxidative damage to DNA thus reproducing a senescent-like profile of sensitivity. In turn, DNA from old cultures incubated in a NAD precursor-free medium was more prone to H2O2-induced strand breaks mimicking DNA sensitivity of young cells. The extent of oxidatively-induced DNA damage in MRC5 populations correlated inversely with the levels of glutathione peroxidase (GPx) activity that almost doubled when cells passed from the young to the senescent stage. In addition, H2O2-pretreatment of young cells induced an increase in GPx expression approaching old cell values and promoted also the premature appearance of neutral beta-galactosidase activity and decreased c-fos expression upon serum stimulation, both of which were assumed to be characteristic traits of the senescent phenotype.

Interaction of urokinase-type plasminogen activator with its receptor rapidly induces activation of glucose transporters.

The interaction of urokinase-type plasminogen activator (u-PA) or of u-PA amino-terminal fragment (u-PA-ATF) with the cell surface receptor (u-PAR) was found to stimulate an increase of glucose uptake in many cell lines, ranging from normal and transformed human fibroblasts, mouse fibroblasts transfected with human u-PAR, and cells of epidermal origin. Such increase of glucose uptake reached a peak within 5-10 min, depending on the cell line, and occurred through the facilitative glucose transporters (GLUTs), since it was inhibited by cytochalasin B. Each cell line showed a specific mosaic of glucose transporter isoforms, GLUT2 being the most widespread and GLUT1 the most abundant, when present. u-PAR stimulation was followed by translocation of GLUT1 from the microsomal to the membrane compartment, as shown by both immunoblotting and immunofluorescence of sonicated plasma membrane sheets and by activation of GLUT2 on the cell surface. Both translocation and activation resulted inhibitable by protein-tyrosine kinase inhibitors and independent of downregulation of protein kinase C (PKC). The increase of intracellular glucose was followed by neosynthesis of diacylglycerol (DAG) from glucose, as previously shown. Such neosynthesis was completely inhibited by impairment of facilitative GLUT transport by cytochalasin B. DAG neosynthesis was followed by activation of PKC, whose activity translocated into the intracellular compartment (PKM), where it probably phosphorylates substrates required for u-PAR-dependent chemotaxis. Our data show that u-PAR-mediated signal transduction, related with u-PA-induced chemotaxis, involves activation of tyrosine kinase-dependent glucose transporters, leading to increased de novo DAG synthesis from glucose, eventually resulting in activation of PKC.

Induction, effects, and quantification of sublethal oxidative stress by hydrogen peroxide on cultured human fibroblasts.

Conditions to induce and parameters to evaluate sublethal oxidative stress of cultured human fibroblasts have been investigated in the attempt to identify markers for a more accurate quantification of cell injury. Sublethal oxidative stress was obtained by treating fibroblasts with 0.5 mM H2O2 in DMEM plus 5% FCS for times not exceeding 60 min. Under these conditions cells remained viable throughout long-term incubation, showing no appreciable release of cytosolic enzymes into the medium. On the contrary, exposures of fibroblasts to 0.5 mM H2O2 for times > 60 min induced a lethal cell injury which was fully expressed 2 days later by massive monolayer wasting and leakage of cytosolic components. Early metabolic effects of sublethal stress consisted of a rapid and significant fall of both ATP and NAD+ pools. Concomitantly, there was a moderate increase (about threefold) in both ADP-ribosyl transferase activity and free [Ca2+]i, while the specific activity of glyceraldehyde-3-phosphate dehydrogenase was partially decreased upon treatment. Oxidative injury also caused delayed effects consisting of a large depression of both protein and DNA synthesis. However, while the former was partially restored within 10 days of incubation, the latter remained severely impaired, as encountered in a growth-arrested population. Microfilaments of H2O2-treated cells appeared to be morphologically altered due to partial fragmentation of cytoskeleton actin which, however, was still maintained in the polymerized form as F-actin. Moreover, sublethally injured fibroblasts exhibited a reduced adhesiveness to plastic once they were detached and reseeded into new dishes. Relative adhesion efficiencies (number of adherent cells at 16 h as a percentage of seeded cells) were found to correlate inversely with times of exposure to H2O2. This finding allowed the identification of a biological parameter which showed itself to be very sensitive to oxidative stress and was also useful for developing an assay to grade sublethal injury to fibroblasts.

Production of second messengers following chemotactic and mitogenic urokinase-receptor interaction in human fibroblasts and mouse fibroblasts transfected with human urokinase receptor.

We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transfected with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca2+ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG threshold level required to trigger DNA synthesis.

Changes in pyridine and adenine nucleotide levels in Friend erythroleukaemia cells during growth and differentiation.

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.

Role of specific membrane receptors in urokinase-dependent migration of human keratinocytes.

On the basis of both 125I-labeled plasminogen activator binding analysis and transmission electron microscopy studies of the interaction of a plasminogen activator/gold complex with cell membranes, we have found that human keratinocytes have specific receptors for human urokinase-type plasminogen activator distributed on the cell surface as singlets, or as small or large clusters. The use in binding experiments of the purified A chain of urokinase-plasminogen activator and of anti-A chain monoclonal antibodies has indicated that cell receptors are specific for a sequence present on the A chain, as previously reported for other cells. The interaction of both the native molecule and the purified A chain with such receptors stimulates mobilization of keratinocytes in an in vitro cell model system (Boyden chamber), when present in the lower compartment of the migration apparatus in nanomolar concentrations. Preincubation of chemoattractants with a monoclonal antibody which prevents receptor/ligand interaction also prevents plasminogen activator-induced cell migration. These data suggest that, under the conditions used in this in vitro model system, the plasminogen activator-dependent mobilization of keratinocytes depends on the interaction of the ligand with free receptors on the cell surface, and is independent of plasmin generation.

Modulation of urokinase receptors on human synovial cells and osteoarthritic chondrocytes by diacetylrhein.

On the basis of both 125I-labelled urokinase-like plasminogen activator binding analysis and transmission electron microscopy of an urokinase-gold complex, we have shown the presence of specific receptors for human urokinase on the cell membrane of human synovial cells. By radio-ligand binding experiments we have shown the existence of similar receptors on the surface of human chondrocytes. In both cases the specific binding is attributable to interaction between the receptor and the A chain of the ligand, as previously shown in other cell model systems. Treatment of synoviocytes with 1,8-diacetoxy-anthraquinone-3-carboxylic acid (diacetylrhein) is able to reduce the number of surface urokinase receptors. At the same time the drug can reduce the fibrinolytic activity released into the culture medium of human synovial cells. Preliminary data indicate that chondrocytes from osteoarthritic patients have a larger number of urokinase receptors than chondrocytes of normal patients. Diacetylrhein can restore the receptor number to normal levels; the amount of urokinase in the synovial fluid of ostroarthritic patients is also reduced. We conclude that the use of this drug has the chance to significantly modify the natural history of osteoarthritis.

Inhibition of spontaneous growth and induced differentiation of murine erythroleukaemia cells by paraquat and atrazine.

The effect of the herbicides paraquat and atrazine on erythroid differentiation has been studied in mouse erythroleukaemic cells. The addition of atrazine or paraquat was shown to inhibit both spontaneous growth and hexamethylene-bis-acetamide (HMBA)-induced differentiation of undifferentiated erythroleukaemic cells. This effects was dose-dependent and occurred at concentrations of less than 10 ppm for both herbicides. Growth inhibition with atrazine (40-45%) was less pronounced than with paraquat (85-90%). Inhibition of differentiation paralleled growth inhibition. A synergistic effect was observed with HMBA, which per se reduced the growth rate of mouse erythroleukaemic cells, and either herbicide. Evaluation of cell viability under all the experimental conditions using either a trypan blue dye exclusion test or labelled chromium indicated that the effects observed were not related to a cytocidal action of atrazine or paraquat.

Plasminogen activators and tiaprofenic acid in inflammation. A preliminary study.

Treatment with tiaprofenic acid appreciably reduced the level of plasminogen activators in the medium of 3T3-Balb mouse fibroblasts, as revealed by both a fibrin plate assay and amidolytic determination with chromogenic substrates. At the same time, tiaprofenic acid was able to inhibit the production of plasminogen activators induced by phorbol myristate acetate, a powerful inflammation and tumour promoter, added to the cell monolayers. By isolating the inhibitors of plasminogen activators it was possible to show that the decrease of fibrinolytic activity produced by tiaprofenic acid is not related to an increase of inhibitors. Rather, a decrease of activators seems to take place. Synovial fluid samples from 4 patients before and after treatment with tiaprofenic acid were also assayed for plasminogen activator activity by the fibrin lysis method. In 3 of the 4 cases a marked decrease after treatment was evident. The one unresponsive patient suffered from a para-neoplastic arthritis.

Relationship between pyridine nucleotide levels and ribonucleotide reductase activity in Yoshida ascites hepatoma AH130.

We measured both pyridine nucleotide levels and ribonucleotide reductase-specific activity in Yoshida ascites hepatoma cells as a function of growth in vivo and during recruitment from non-cycling to cycling state in vitro. Oxidized nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADP) levels remained unchanged during tumour growth, while NADP+ and reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels were very high in exponentially growing cells and markedly decreased in the resting phase. Ribonucleotide reductase activity paralleled NADP(H) (NADP+ plus NADPH) intracellular content. The concomitant increase in both NADP(H) levels and ribonucleotide reductase activity was also observed during G1-S transition in vitro. Cells treated with hydroxyurea showed a comparable correlation between the pool size of NADP(H) and ribonucleotide reductase activity. On the basis of these findings, we suggest that fluctuations in NADP(H) levels and ribonucleotide reductase activity might play a critical role in cell cycle regulation.

Effects of liver regeneration on tRNA contents and aminoacyl-tRNA synthetase activities and sedimentation patterns.

The tRNA content and aminoacyl-tRNA synthetases of regenerating liver in the phase of rapid growth were compared with those of livers from both intact and sham-operated rats. At 48 h after hepatectomy, the amount of active tRNA (called 'total acceptor capacity') is significantly higher in regenerating liver than in control livers, owing to a general, possibly not uniform, increase in the various tRNA families, which suggests that it may contribute to the increased protein synthesis and to decreased protein degradation as well. The activities of most, but not of all, aminoacyl-tRNA synthetases in cell sap of regenerating liver tend to be greater than normal. Increased activity of histidyl-tRNA synthetase fits in with the possibility that the mechanisms that control the rate of protein degradation through aminoacylation of tRNAHis in cultured cells [Scornik (1983) J. Biol. Chem. 258, 882-886] also operate in the liver and play a role in regeneration. Sedimentation analysis of cell sap in sucrose density gradients shows a shift of prolyl-tRNA synthetase activity toward the high-Mr form in regenerating liver. This change might be related to the positive protein balance and to growth in vivo, since it is also observed in the anaplastic Yoshida ascites hepatoma AH 130.

Metabolic aspects of cell cycle regulation in normal and cancer cells.

Several studies are reviewed dealing with the mechanisms which regulate the cell cycle progression in normal and cancer cells. Using Yoshida AH 130 ascites tumor cells, it has been found that the G1-S transition of these cells is impaired by specific inhibitors of the electron flow through the respiratory chain (antimycin A), although respiratory ATP can be replaced by glycolytic ATP. The above transition can be also inhibited by the addition of physiologic substrates, mainly pyruvate, by a mechanism which appears linked to a modification of the cellular redox state and can be totally reversed by adding adenine to the culture medium. Adenine equally removes the block produced by antimycin A, pointing out a respiration-linked step of purine metabolism restricting the cell recruitment into S. A substantial protection of this step against the inhibitory effects of pyruvate and antimycin A has been obtained by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked limiting step of tumor cell cycling involves folate metabolism and its connection to purine synthesis. The biologic relevance of these findings is stressed by the fact that pyruvate addition also inhibits the proliferation of concanavalin A-stimulated lymphocytes as well as of bone marrow hemopoietic cells in the presence of colony-stimulating factors. On the other hand, pyruvate only slightly affects the growth kinetics of malignant lymphoblasts and of Friend erythroleukemia cells either in the absence or in the presence of the differentiation inducer dimethylsulfoxide.

The respiration-linked limiting step of tumor cell transition from the non-cycling to the cycling state: its inhibition by oxidizable substrates and its relationships to purine metabolism.

The recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G1-S transition of these cells, unaffected by 2,4-dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption. Addition of suitable concentrations of preformed purine bases (mainly adenine), completely removes the block of the G1-S transition produced either by the excess of oxidizable substrates or by antimycin A. These findings indicate the existence of a respiration-linked step in purine metabolism, which restricts the above transition and is equally impaired by blocking the respiratory chain or by saturating it with an excess of reducing equivalents derived from unrelated oxidations. The inhibitory effects of pyruvate and antimycin A can be largely removed by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked restriction point of tumor cell cycling involves the folate metabolism and its connections to purine synthesis.

[Multivariate analysis of aminoacyl-tRNA synthetase activities of liver and hepatomas (author's transl)]. / Analisi multivariata delle attività aminoacil-tRNA sintetasiche del fegato e degli epatomi

Data on the aminoacyl-tRNA synthetase activities in normal liver and in three transplantable rat hepatomas, viz Yoshida's AH 130 and Morris's 5123C and 7793, were studied by means of factor analysis, a powerful technique of multivariate analysis particularly suitable foridentifying factors which theoretically may account for the pattern of interrelations between several variables. The analysis has been performed on 51 patterns of enzyme activities, covering 17 out of the 20 aminoacyl-tRNA synthetases; 80% on average of the total variability of the 17 enzyme activities may be accounted for by the first 4 factors extracted. The enzymes seem to fall into different groups, depending on their relationship with the factors thus identified. The results suggest that enzymes belonging to the same group share a common control mechanism, and are independent of the enzymes belonging to different groups, both in normal liver and in hepatomas.