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.

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.

Characterization of human buccal epithelial cells transfected with the simian virus 40 T-antigen gene.

Serum-free cultures of normal human buccal epithelial cells were transfected with a plasmid containing the SV40 T-antigen (SV40T) gene. Two major lines developed that showed extended lifespans (between 30 and 40 weeks) as compared with the controls (approximately 6 weeks). Continued growth through one or two crises generated several sublines. They expressed the epithelial marker keratin and also exhibited nuclear expression of SV40T. The lines showed abnormal karyotypes with both numerical and structural aberrations and variably responded to agents that normally inhibit growth and/or induce terminal differentiation, i.e. transforming growth factor-beta 1 and fetal bovine serum. One of the lines, termed SVpgC2a, developed into an apparently immortal line, since it had undergone more than 700 population doublings from over 2 years in culture. Further characterization of this line demonstrated its clonal origin, with integration of two copies of SV40T at the same site and the presence of both normal retinoblastoma and wild-type p53 proteins. This line showed high resistance to growth inhibition by transforming growth factor-beta 1 and serum similar to that shown by buccal carcinoma cell line SqCC/Y1. Neither SVpgC2a nor its parental lines were tumorigenic when injected into athymic nude mice, whereas the SqCC/Y1 cells induced tumors. The various lines with extended but finite lifespans, complemented by one immortalized line, which retained non-malignant properties upon extended culture, provide a battery of model systems that will be useful for studying mechanisms of human oral carcinogenesis.

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.

Structure, stability and biological properties of a N-terminally truncated form of recombinant human interleukin-6 containing a single disulfide bond.

A mutant species of the 185-residue chain of human interleukin-6 lacking 22-residues at its N-terminus and with a Cys-->Ser substitution at positions 45 and 51 was produced in Escherichia coli. The 163-residue protein des-(A1-S22)-[C45S, C51S]interleukin-6, containing a single disulfide bridge, formed inclusion bodies. Mutant interleukin-6 was solubilized in 6 M guanidine hydrochloride, subjected to oxidative refolding and purified to homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The purity of the mutant species was established by electrophoresis, isoelectrofocusing and reverse-phase HPLC and its structural identity was checked by N-terminal sequencing of both the intact protein and several of its proteolytic fragments. Electrospray mass spectrometry analysis of mutant interleukin-6 gave a molecular mass of 18,695 +/- 2 Da in excellent agreement with the calculated value. Circular dichroic, fluorescence emission and second-derivative ultraviolet absorption spectra indicated that mutant interleukin-6 maintains the overall secondary and tertiary structure, as well as stability characteristics, of the recombinant wild-type human interleukin-6. The urea-induced unfolding of mutant interleukin-6, monitored by circular dichroic measurements in the far-ultraviolet region, occurs as a highly cooperative process with a midpoint of denaturation at 5.5 M urea. The data of the reversible unfolding of mutant interleukin-6 mediated by urea were used to calculate a value of 20.9 +/- 0.4 kJ.mol-1 for the thermodynamic stability of the protein at 25 degrees C in the absence of denaturant. The biological activity of mutant interleukin-6 was evaluated in vitro by the hybridoma proliferation assay, and in vivo by measuring thrombopoiesis in monkeys. Dose/response effects of the mutant were comparable or even higher than those of the wild-type protein. Overall the results of this study show that mutant interleukin-6 is a biologically active cytokine, which could find practical use as a therapeutic agent.

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.

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.

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).

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.

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.

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.

Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues.

The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis. Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.

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.

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.

Serum-free growth and karyotype analyses of cultured normal and tumorous (SqCC/Y1) human buccal epithelial cells.

Epithelial cell cultures were obtained following tryptic digestion of normal human buccal mucosa. Primary cultures exhibited markedly higher colony-forming efficiencies and growth rates using fibronectin/collagen-coated, as compared to non-coated culture dishes and a serum-free MCDB 153 medium developed for epidermal epithelial cells than a similar medium previously developed for buccal explant outgrowth cultures. At the preferred conditions, the cells could be transferred at least 5-fold, divided at about one population doubling per day, and commonly underwent 60 population doublings resulting in yields of 10(8) to 10(11) cells per cm2 mucosal specimen. Moreover, these conditions successfully cultivated a buccal carcinoma cell line (SqCC/Y1) for several months. The carcinoma cells were resistant to factors that inhibited growth or induced differentiation of normal cells, i.e., transforming growth factor type beta 1, Ca2+, or serum. Karyotype analyses of SqCC/Y1 cells showed 63 to 83 chromosomes per metaphase and consistent occurrences of monosomy 1, tetrasomy 19 and 20, as well as trisomy 22, and at least 7 marker chromosomes, whereas cells obtained from non-cancerous donors were diploid. It is concluded that the similarly defined culture conditions may now be applied to study characteristics of both normal and tumorous buccal epithelial cells.

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.

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.

Human ovarian tumors in primary culture: growth, characterization and initial evaluation of the response to cis platinum treatment in vitro.

A novel cell culture system is reported for the growth of ovarian tumors. Two approaches were developed to isolate tumor cells, one for ovarian carcinomas and the other for benign cystomas or borderline cystadenomas, which yield virtually pure tumor-cell clusters. The plating efficiency exceeded 10% in approximately 80% of the processed surgical specimens. Cells grown in a newly developed KOV medium (a modification of MCDB 151 supplemented with 6 defined growth factors and a moderate amount of FBS) had an average growth rate of 0.23 population doublings/day. Primary tumor-derived cultures, including those derived from cystomas, were analyzed by flow cytometry demonstrating a DNA heteroploid content in 55% of the cases. The neoplastic origin of the cells in culture was further confirmed by 3 monoclonal antibodies (OC125; MOv2; MOv19) with high specificity against epithelial ovarian malignancies. Cultures were tested with cis-DDP to determine their suitability for pharmacological studies. Exposure to the drug (from 10 to 80 microM for 1 hr) resulted in variable cell-killing responses, and the prominent effect on cell-cycle progression in primary cultures was a prolonged arrest in S phase. The formation and persistence of DNA-ISC caused by an exposure to 40 microM cis-DDP for 1 hr was studied by alkaline elution in 6 different tumor-derived cultures. DNA-ISC equivalents were highest between 9 and 24 hr after treatment and were repaired only to a limited extent within 48 hr of recovery time. The present study confirms the usefulness of this culture system for pharmacological studies of active chemotherapeutic agents against human ovarian tumors.

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.