Suramin modulates cellular levels of hepatocyte growth factor receptor by inducing shedding of a soluble form.

Several growth factor receptors undergo shedding from the cell surface as a result of limited proteolysis via mechanisms that are at present poorly understood. By Western blotting of the conditioned media and cell lysates of several cell lines expressing the hepatocyte growth factor receptor, we found that suramin, a pharmacological agent that inhibits the activity of many growth factors, was able to induce shedding of this receptor. Increased levels of soluble hepatocyte growth factor receptor were observed in the conditioned media of GTL-16, a cell line over-expressing the receptor, as early as ten minutes after initial exposure to the agent, and incubation of this line with 300 microM suramin caused a 50% reduction in cell-associated levels of receptor after 6 hours. Although protein kinase C activation by treatment of cells with phorbol esters has previously been found to stimulate shedding of the hepatocyte growth factor receptor, this hitherto undescribed activity of suramin was not affected by protein kinase C inhibitors. Since shedding represents a possible means of down-modulation of receptor activity, suramin may inhibit the hepatocyte growth factor ligand/receptor system, not only by abrogation of hepatocyte growth factor binding to intact receptor, but also by induction of receptor shedding.

Preclinical and clinical studies with recombinant human basic fibroblast growth factor.

Basic fibroblast growth factor is a polypeptide belonging to a family of natural proteins also known as heparin-binding growth factors endowed with a pleiotropism of biological activities, the most striking of which are related to wound healing. Large quantities of recombinant human basic fibroblast growth factor (rh-bFGF) of a clinical grade were obtained and used to undertake preclinical and clinical studies. In vivo the wound healing effect of rh-bFGF was evaluated in experimental targets such as the cornea and the tympanic membrane, showing a significantly increased epithelial healing rate in drug-treated animals. The deposition of labeled rh-bFGF after topical applications in ocular wounding models did not result in a systemic absorption of the intact rh-bFGF molecule. The acute and the subchronic toxicity studies undertaken after iv and topical administration of a stable pharmaceutical formulation of rh-bFGF did not result in irritation, and no signs of general toxicity were observed. Altogether these data permitted us to start recently with human studies, which are still ongoing, aimed to evaluate the tolerability and the activity of rh-bFGF on tegumental targets such as the cornea and the skin.

Alanine scan of endothelin: importance of aromatic residues.

A systematic approach to map the functional important determinants of endothelin-1 (ET) by an alanine scan is described. Studies on the in vitro receptor binding affinity and on the agonist contracting activity defined that residues Asp8, Tyr13, Phe14, Leu17, and Trp21 were of major biological significance. A striking observation was that four out of these five sites were hydrophobic amino acids. Ala analogues of the aromatic residues at position 13, 14, and 21 displayed sharply reduced receptor binding affinity (< 2% of ET) and can be considered important for receptor contact. Ala analogues of Asp8 and Leu17 lost most (> 90%) of the agonist activity but retained a receptor affinity nearly equivalent to ET and can be considered to be important for signal transduction. Three other positions, Val12, Asp18, and Ile20 (which are adjacent to the biologically important sites of Tyr13, Leu17, and Trp21), resulted as partially tolerant to Ala substitution, retaining 14-50% of the potency of ET. Ala analogues of the Et isomeric disulfide arrangement (Cys1,11 and Cys3,15) were always less active than the corresponding analogues with the native disulfide pairings (Cys1,15 and Cys3,11).

Production of homogeneous basic fibroblast growth factor by specific enzymatic hydrolysis of larger microheterogeneous molecular forms.

The 146-amino acid form of basic fibroblast growth factor (bFGF) was expressed in Escherichia coli and purified by a two step process including ion exchange and heparin-Sepharose chromatographies. However, the resulting protein consisted of a mixture of 146- and 145-amino acid forms, indicating that, besides the initial methionine, also the following residue (proline) was removed from the N-terminus. The same phenomenon was observed when the 155-amino acid form, which is biologically equivalent to the shorter one, was expressed in E. coli. Taking into account the previously known data concerning the possible mechanism of cleavage of the extended forms of bFGF in vivo, we developed an efficient enzymatic process that allows the production of an homogeneous 146-amino acid form from recombinant NH2-end extended forms. This process takes advantage of the protecting effect that heparin exerts on bFGF. Accordingly, when bFGF, complexed to heparin, is treated with pepsin A, an aspartic protease with a broad specificity, only the Leu9-Pro10 peptide bond is cleaved generating the 146-amino acid form. Quantitative yields of this reaction are also achieved when bFGF is bound to a heparin-Sepharose column, allowing the integration of this enzymatic step directly during purification of the recombinant extended forms of bFGF.

Biotransformation and intracellular binding of arsenic in tissues of rabbits after intraperitoneal administration of 74As labelled arsenite.

The fate of arsenite was studied in rabbits injected i.p. with 1 microgram As/kg body wt as 74As labeled AsO2-. Eight tissues plus plasma and urine were analyzed for 74As content at different times. Arsenic was rapidly metabolized and poorly retained in the tissues. The main metabolite present in urine and plasma was dimethylarsinic acid. Sixty percent of the dose was excreted via urine and 6% with feces during the first day. In plasma arsenic was present mainly in a diffusible form, showing a very poor binding affinity to plasma proteins. Chromatographic separations and membrane ultrafiltrations showed that in liver and kidney cytosols, arsenic was significantly associated to proteins. The diffusible fraction disappeared within 48 h. The fraction of arsenic bound to proteins was suggested to be inorganic arsenic whereas the methylation process was closely related to the elimination and the detoxification of inorganic arsenic.

Relationships between iron and vanadium metabolism: the exchange of vanadium between transferrin and ferritin.

The study of possible relationships between iron and vanadium metabolism (E. Sabbioni and E. Marafante, Proc. XIth Int. Conf. Biochem., 13-5-R122, Toronto, Canada) was extended to the vanadium in the biochemical mechanisms which involve the exchange of iron between transferrin and ferritin. The transfer of vanadium between transferrin and ferritin was investigated using 48V radiotracer and gel filtration technique. 48V labeled human transferrin and horse spleen ferritin, 48V plasma from rats injected with 48VO2+, unlabelled rat liver cytosol, and plasma were used as sources of the two proteins for their incubation under different conditions. The results show that the equilibrium: V - transferrin in equilibrium V - ferritin occurs in vitro at physiological pH under the conditions of this experiment. No transfer of vanadium between the two proteins, however, occurs when they are incubated simply in a buffer at pH = 7.4. The maximum transfer was observed when transferrin and ferritin were mixed in their natural environments such as plasma and liver cytosol. This suggests that the exchange of the vanadium between the two proteins is affected by biochemical factors which are present in the body. A brief evaluation of the significance on the very low amounts of the element exchanged between the two proteins is also presented.

Experience with the preclinical assessment of basic fibroblast growth factor (bFGF).

Repeated intravenous administrations were carried out in cynomolgus monkeys and rats (S.D.) for a maximum of 4 weeks at doses of 1, 10 and 100 micrograms/kg/day in stable formulation. Three main target organs were identified: red blood cells (RBC), kidney glomeruli (KG) and bone at the top dose level. RBC: Normochromic normocytic anaemia started in rats and monkeys during the second week of treatment (decrease in red blood cell production). The kinetics of this anaemia, as well as its recovery, will be discussed. Bone: Dramatic hyperostosis in rats was present by day 10 in long or spongious bone. This became marked on day 29 and regressed after treatment was stopped. KG: In the rat glomerular lesions were present starting from day 16. They consisted of enlargement and vacuolation of podocytes with loss of foot processes and adhesions between glomerular tuft and Bowman's capsule. Proteinuria was a striking feature. In the monkey the lesions were hyperplasia of the parietal epithelium of Bowman's capsule which involved replacement of normally flattened epithelium by cuboidal cells, with some pseudostratification. Proteinuria also occurred in monkeys, accompanied by a lowering of serum protein (albumin). In two animals, death (by day 15) was preceded by high levels of urea and blood creatinine. The above lesions (KG) disappeared almost completely over a recovery period. It is suggested that these phenomena are not the expression of direct toxicity in the form of lethal insults, but rather a manifestation of a change in cell activity.

Intracellular interaction and biotransformation of arsenite in rats and rabbits.

The accumulation of arsenic with time in tissues of rats and rabbits was determined by neutron activation analysis (NAA). Rats showed a steady increase in the As-concentrations with age, whereas in rabbits it was nearly the same for adults and in young animals. The metabolism of arsenic was studied in both animal species after i.p. injection of 50/micrograms As/kg b.w. as 74As labelled arsenite. Eight tissues, as well as blood and urine, were analysed for 74As content after 16 and 48 hours. The binding of 74As to hematic and intracellular components and the chemical forms of arsenic in tissues and urine were investigated. In the plasma and the RBC-fraction of the rabbit, the As concentration decreased during the first two days, while in the rats it only disappeared from the plasma, but was retained in the RBC-fraction. Liver, kidney and lung of rabbits with the highest As concentrations at 16 and 48 hours showed a rapid clearance of As in the first 48 hours. In the corresponding tissues of the rats, the rate of decline was significantly lower, due to the higher binding of 74As to tissue constituents. Poor binding of As to plasma proteins was seen in rabbits while in rats it was totally bound to this fraction. In the RBC, liver and kidney cytosols, however, the affinity of As for intracellular proteins was higher in both animal species but characterized by a rate of binding different between the two animal species. The amount of dimethylarsinic acid (DMA) in the tissues was significantly lower in the rat than in the rabbit, reflecting the total amount of diffusible arsenic, which was also much lower in the tissues of rats than in rabbits.

Neoplastic transformation of BALB/3T3 cells by metals and the quest for induction of a metastatic phenotype.

In the mouse embryo cell line BALB/3T3 Clone A31-1-1, dose-dependent morphologic neoplastic transformation was obtained with NaAsO2, Na2HAsO4, CdCl2, and K2CrO4. Cellular uptake was four fold higher for As3+ than for As5+, and As5- was metabolized to As3+ in cytosol. Cytotoxicity and transformation rates were four fold higher for As3+ than As5+, but when correlated to cellular As burden they were equivalent. As3+ appears responsible for the transforming activity. The foci transformed by metals (or by other carcinogens) gave rise to tumorigenic cell lines (sc sarcomas in nude mice), none of which, however, induced metastases when tested by sc or by iv injection in nude mice. Thus carcinogens change this aneuploid cell line from a preneoplastic stage to the expression of malignant growth but not of metastatic activity. Metastatic and type IV collagenolytic activities can be induced by transfection of the c-Ha-ras oncogene and inhibited by the Ad2-E1a gene (so far shown in other cell types). It remains to be seen whether metal or other carcinogens can induce the nonmetastatic phenotype to become metastatic. The molecular mechanisms of metal carcinogenesis, studied in cell culture systems, in combination with other factors or oncogenes, may reveal the effect of individual metal carcinogens on discrete steps of the complex process of carcinogenesis.

An immunodominant epitope in a functional domain near the N-terminus of human granulocyte-macrophage colony-stimulating factor identified by cross-reaction of synthetic peptides with neutralizing anti-protein and anti-peptide antibodies.

We produced polyclonal and monoclonal antibodies (MAbs) against recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and performed studies of epitope mapping by ELISA, using five synthetic peptides corresponding to sequences along this molecule. Additionally, anti-peptide MAbs were generated. The antibody ability to inhibit rhGM-CSF activity was determined using as bioassay the MO7e cell line, which is dependent on hGM-CSF for growth in vitro. An immunodominant epitope able to induce the highest neutralization antibody titers was identified near the N terminus of hGM-CSF. A synthetic peptide 14-24, homologous to a sequence including part of the first alpha-helix of the molecule, was recognized by neutralizing anti-protein antibodies. Similarly, MAbs anti- 14-24 cross-reacted with rhGM-CSF and specifically blocked its function. Replacement of Val16 or Asn17 with alanine greatly reduced the antibody-binding capacity to peptide 14-24, whereas substitution of Gln20 or Glu21 was less critical. Monoclonal antibodies generated against residues 30-41 (corresponding to an intrahelical loop) and 79-91 (homologous to a sequence including part of the third alpha-helix) or its analog [Ala88](79-91)beta Ala-Cys, were conformation dependent and nonneutralizing: they failed to react or bound poorly to rhGM-CSF in ELISA, but readily recognized the homologous sequence in the denatured protein, by Western blotting.

Bombesin receptor antagonists. 5 new irreversible alkylating analogues.

New alkylating bombesin analogues were synthesized in order to increase their solubility and stability in aqueous solutions. The best compromise between these parameters and the biological properties (receptor binding and antagonistic activity) was achieved with 4-[bis(2-chloro-ethylamino)]benzoyl derivatives of the BN (7-14) octapeptide carrying a (13-14) reduced peptide bond independently of the presence of a His12 residue, either free or protected.

Changes in renal handling of platinum in cisplatinum-treated rats following induction of metabolic acidosis or alkalosis.

Manipulations of metabolic acid-base status were used in an attempt to modify the renal handling and toxicity of the anticancer drug cisplatinum. Rats were orally pretreated with either tap water (TW), ammonium chloride (AC), or sodium bicarbonate (SB) for 3 days prior to intraperitoneal administration of a high cisplatinum dose (7.5 mg/kg b.w.). Urine was collected daily for 4 days between drug dosing and killing of the animals. AC-pretreated rats did not exhibit the characteristic cisplatinum-induced diuresis and were unable to maintain an acid urinary pH following drug administration. AC rats had a significantly lower, and SB rats a significantly higher, urinary excretion of platinum than did TW rats. Platinum excretion was found to be correlated with urinary pH (r = 0.88) and not urinary volume (r = 0.30). The renal concentration of platinum was greater in AC animals than in SB or TW animals, but no significant difference was observed in liver or plasma concentrations between the groups. Both pretreated groups had equal percent of free vs bound platinum. Proteinuria was more severe in AC-pretreated rats, but histologic evidence of renal tubular damage was present in all the three groups. It is concluded that metabolic acidosis can seriously impair the renal handling of high dose cisplatinum but that metabolic alkalosis offers no evident advantages over nonpretreatment.

Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation.

Cytotoxicity, morphological neoplastic transformation, cellular uptake and metabolic reduction were determined in BALB/3T3 Cl A31-1-1 cells for trivalent arsenic (sodium arsenite, As3+) and for pentavalent arsenic (sodium arsenate, As5+). The levels of cellular uptake of 73As-labelled sodium arsenite and arsenate were dose-dependent and highest in the first hour. At equimolar concentration (3 X 10(-6) M), cellular uptake was 4-fold higher for As3+ than for As5+. Cytotoxicity was higher for As3+ than for As5+, but when correlated to total As cell burden it showed no significant difference for the two forms. Morphological transformation focus assays showed transforming activity for both As3+ and As5+, with relative transformation frequencies also of approximately 4:1. Recovery from the cytosol after exposure for 1-24 h was greater than 90% for either form of absorbed As. Exposure to As3+ yielded 100% as As3+ in cytosol, but exposure to As5+ yielded greater than 70% as As3+, showing a high rate of intracellular metabolic reduction. No methylated metabolites were detected by ion-exchange chromatography. After 24-h incubation in cell-free medium, oxidation of As3+ to As5+ occurred up to 30% of the dose, but incubation in the presence of cells lowered the oxidation level to 4%. As5+ was recovered unchanged from cell-free medium (24-h incubation), but in the presence of the cells it yielded up to 5% as As3+ within 24 h and the cumulative release of As3+ by cells exposed to As5+ was dose-dependent. Glutathione depletion by diethylmaleate inhibited reduction of As5+ to As3+ by these cells up to 25% of controls, showing that As5+ reduction is partly dependent on glutathione. These results suggest that As3+ is the form responsible for the cytotoxic and transforming effects, independently of the valence state of the inorganic arsenic in the culture medium.

Endothelin-1-selective binding sites are downregulated by transforming growth factor-beta and upregulated by basic fibroblast growth factor in a vascular smooth muscle-derived cell line.

Endothelins (ETs) elicit in vivo and in vitro a potent vasoconstrictor activity after binding to high-affinity receptors on vascular smooth muscle cells (VSMC). A617 cells, a VSM-derived cell line, were used as an in vitro model system to study selected growth factors and cytokines involved in proliferative and/or inflammatory diseases of the vessel wall as possible regulators of the high-affinity binding capacity of ET-1 to the cells. Radioligand studies characterized the binding of ET-1 to the isopeptide selective ETA receptor subtype on A617 cells as a time- and temperature-dependent saturable process (Kd = 0.13 +/- 0.04 nM, Bmax = 49 +/- 7 fmol/10(6) cells). Pretreatment of A617 cells with basic fibroblast growth factor (bFGF), a mitogenic agent for vascular cells, resulted in a time- and dose-dependent increase in ET-1 binding capacity, whereas preexposure to transforming growth factor-beta (TGF-beta) induced a reduction of the Bmax for ET-1. Platelet-derived growth factor (PDGF), interleukin-6 (IL-6), tumor necrosis factor-alpha, and fetal bovine serum (FBS) pretreatments did not affect consequent ET-1 binding to A617 cells.

Differential effects of leukotriene C4 on endothelin-1 and prostacyclin release by cultured vascular cells.

Nanomolar concentrations of leukotriene C4 and phorbol 12-myristate acetate, a protein kinase C activator, stimulated endothelin-1 release by vascular endothelial but not smooth muscle cells. For both agonists, attenuation of this stimulatory effect was observed at higher concentrations, concomitant with but independent of enhanced prostacyclin biosynthesis.

Basic fibroblast growth factor stimulates hepatocyte growth factor/scatter factor secretion by human mesenchymal cells.

Basic fibroblast growth factor (bFGF) together with other pleiotropic factors plays an important role in many complex physiological processes such as embryonic development, angiogenesis, and wound repair. Among these factors, hepatocyte growth factor/scatter factor (HGF/SF) which is secreted by cells of mesodermal origin exerts its mito- and motogenic activities on cells of epithelial and endothelial origin. Knowledge of the regulatory mechanisms of HGF/SF may contribute to the understanding of its role in physio-pathological processes. We observed that the secretion of HGF/SF by MRC-5 cells and by other fibroblast-derived cell cultures in conditioned media was enhanced by exposure to bFGF. HGF/SF was measured by the scatter assay, a bioassay for cell motility, and was further characterized by Western blot analysis with anti-HGF/SF antibodies. Exposure of MRC-5 cultures to 10 ng/ml of bFGF resulted already 6 h posttreatment in a threefold higher amount of scatter factor secreted into the medium as compared to untreated cultures. HGF/SF secretion was sustained after bFGF treatment for the following 72 h when increased amounts of HGF/SF were detected both in conditioned media as well as associated to the extracellular matrix. The secretion of HGF/SF in cell supernatants increased dose dependently upon treatment with bFGF starting from basal levels of 6 U/ml and reaching 27 U/ml at 30 ng/ml bFGF, plateauing thereafter. Upregulation of HGF/SF by IL-1, already described by others, was confirmed in this study. Based on our findings an articulated interaction can be speculated for bFGF, HGF/SF, and IL-1, e.g., in tissue regeneration during inflammatory processes or in wound healing.

Spontaneous establishment and characterization of mouse keratinocyte cell lines in serum-free medium.

Mouse keratinocytes cultures readily develop into established cell lines without undergoing a "crisis" in a newly-developed serum-free medium, LEP/MK2. LEP/MK2 consists of calcium-free MEM with non-essential amino acids supplemented with 8 factors. Two lines, MK1 and MKDC4, have been isolated and have now doubled more than 400 and 200 times respectively. In MK1 cells, Giemsa banding has revealed significant karyotypic changes as early as the 4th passage, leading to a near-tetraploid karyotype with random loss and gain of individual chromosomes. Minute chromosomes, but no stable markers have been observed. After these initial changes, examination of cultures at several passage levels has shown that the karyotype has remained essentially stable. The MKDC4 line, also sub-tetraploid at the 7th passage, had 4 marker chromosomes by the 47th passage. The rapid increase in chromosome number may have contributed to the "immortalization" of these lines. The response of these established keratinocyte lines to growth factors and serum-derived inhibitors changed with increasing passage level. Most notable of these changes were a reduction in the requirement for bovine pituitary extract (an absolute requirement for growth of secondary MK1 cells) and a decreased sensitivity to serum and serum-derived inhibitors, e.g., transforming growth factor-beta. The established lines, like primary and secondary keratinocytes, remain responsive to calcium-induced terminal differentiation and are non-tumorigenic in athymic, nude mice. This serum-free system is suitable for transformation studies with oncogenes and chemical carcinogens.

Control of stroma-dependent hematopoiesis by basic fibroblast growth factor: stromal phenotypic plasticity and modified myelopoietic functions.

It has been suggested that basic fibroblast growth factor (bFGF) affects hematopoietic cells directly and that it may also act indirectly by modulating stromal cell functions. We tested the response of phenotypically and functionally distinct stromal cell clones to this cytokine. We studied cell phenotype, the composition and organization of cytoskeleton and extracellular matrix, the ability to repopulate 'wounded areas', the expression of cytokine genes, and the capacity of the stroma to support long-term hematopoiesis in vitro. Although the impact of bFGF on cell growth was small, it induced a prominent morphological change in three stromal cell types that we tested. We analyzed the molecular basis for this change: bFGF modified the protein expression of alpha-smooth muscle actin (alpha-SMA), tropomyosin, alpha-tubulin, fibronectin and paxillin in a distinct manner characteristic of each of the stromal cell types. Immunofluorescence analysis of these proteins revealed profound changes in the cytoskeleton and extracellular matrix (ECM) networks accompanied by increased ability of the 14F1.1 stromal cells to scatter in in vitro 'wounded' areas. Furthermore, although only limited changes were monitored in the expression of cytokine genes, the ability of the stromal cells to support hematopoiesis was markedly modified. Thus bFGF profoundly changes the cellular organization of stromal cells, their adhesion and their motility properties. These changes are associated with modified capacity to support hematopoiesis in culture.