Abstract: Expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) mRNA alternative splice variants was studied in different mouse tissues in hypoxic conditions in vivo. Significant increase of the expression of PFKFB-3 mRNA was observed in the mouse lungs, testes and brain in hypoxia. Several new alternative splice variants of PFKFB-3 mRNA were identified in the lung, testis, brain and skeletal muscle. They have different length and amino acid sequence of C-terminal regulatory part. However, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase catalytic domains were identical. Moreover, the expression of different alternative splice variants of PFKFB-3 mRNA has shown tissue specificity and different levels of induction in hypoxic conditions in vivo. Results of this investigation indicate a possible role of PFKFB-3 splice isoform in cell adaptation to hypoxic conditions.
Abstract: Vascular basement membrane remodeling is involved in tumor angiogenesis to enable tumor invasion and growth. FT-IR spectral imaging was used to determine changes in tumor blood vessels to reveal protein secondary structure in Rag-gamma immuno-deficient mice sacrificed 14 and 21 days after subcutaneous glioma implantation. For the oldest blood capillaries (diameter >20 microns), tumor growth induced a decrease in triple-helix content (1638 cm(-1); -7.3%; P < 0.05) and an increase in beta turns (1666 and 1615 cm(-1); +4%; P < 0.01). These protein-structure alterations, mainly from type IV collagen, reflected the high angiogenic stress of growing tumors. We propose to use these molecular markers of vascular basement membrane protein alterations for gradation of solid tumors by FT-IR spectral imaging.
Abstract: Tumor ischemia participates in angiogenesis and cancer progression through cellular responses to hypoxia and nutrient deprivation. However, the contribution of amino acids limitation to this process remains poorly understood. Using serum-free cell culture conditions, we tested the impact of L-glutamine deprivation on metabolic and angiogenic responses in A549/8 carcinoma cells. In these cells, lowering glutamine concentration modified the cell cycle distribution and significantly induced apoptosis/necrosis. Although glutamine deprivation led to a HIF-independent increase in VEGF-A mRNA, the corresponding protein level remained low and correlated with the inhibition of protein synthesis and activation of the GCN2/eIF2alpha pathway. Limitation of glutamine availability also hampers hypoxia- and hypoglycemia-induced VEGF-A protein upregulation. Thus, glutamine deprivation may have no direct effect on VEGF-dependent angiogenesis, compared to hypoxia or to glucose deprivation, and may instead be detrimental to cancer progression by antagonizing ischemia-induced stresses.
Abstract: In solid tumors, cancer cells subjected to ischemic conditions trigger distinct signaling pathways contributing to angiogenic stimulation and tumor development. Characteristic features of tumor ischemia include hypoxia and glucose deprivation, leading to the activation of hypoxia-inducible factor-1-dependent signaling pathways and to complex signaling events known as the unfolded protein response. Here, we show that the activation of the endoplasmic reticulum stress sensor IRE1 is a common determinant linking hypoxia- and hypoglycemia-dependent responses to the up-regulation of vascular endothelial growth factor-A (VEGF-A). Tumor cells expressing a dominant-negative IRE1 transgene as well as Ire1alpha-null mouse embryonic fibroblasts were unable to trigger VEGF-A up-regulation upon either oxygen or glucose deprivation. These data correlated with a reduction of tumor angiogenesis and growth in vivo. Our results therefore suggest an essential role for IRE1-dependent signaling pathways in response to ischemia and identify this protein as a potential therapeutic target to control both the angiogenic switch and tumor development.
Abstract: Human pancreatic ribonuclease 1 (RNase 1) is a glycoprotein expressed mainly by the pancreas and also found in endothelial cells. The diagnosis of pancreatic cancer (PaC) remains difficult and therefore the search for sensitive and specific markers is required. Previous studies showed that RNase 1 from human healthy pancreas contained only neutral glycans, whereas RNase 1 from PaC cell lines contained sialylated structures. To determine whether these glycan tumor cell-associated changes were also characteristic of serum RNase 1 and could be used as a marker of PaC, we have analyzed the glycosylation of serum RNase 1. The origin of serum RNase 1 was also investigated. Serum RNase 1 from two PaC patients and two controls was purified and the glycans analyzed by high-performance liquid chromatography (HPLC)-based sequencing and mass spectrometry. Although normal and tumor serum RNase 1 contained the same glycan structures, there was an increase of 40% in core fucosylation in the main sialylated biantennary glycans in the PaC serum RNase 1. This change in proportion would be indicative of a subset of tumor-associated glycoforms of RNase 1, which may provide a biomarker for PaC. Two-dimensional electrophoresis of the RNase 1 from several endothelial cell lines, EA.hy926, human umbilical vein endothelial cells (HUVEC), human mammary microvessel endothelial cells (HuMMEC), and human lung microvessel endothelial cells (HuLEC), showed basically the same pattern and was also very similar to that of serum RNase 1. RNase 1 from EA.hy926 was then purified and presented a glycosylation profile very similar to that from serum RNase 1, suggesting that endothelial cells are the main source of this enzyme.
Abstract: Fourier-transform infrared (FT-IR) spectral imaging was used for analyzing biochemical changes in tumor cells. Metabolic parameters of human lung A549/8 adenocarcinoma and U87 glioma cells were compared under stress conditions in culture along with tumor progression after cell implantation onto the chick embryo chorio-allantoic membrane. In cell culture, glucose consumption and lactic acid release were higher in U87 cells. A549/8 cells were less sensitive to oxidative stress as observed through changes in fatty acyl chains. In vivo biochemical mapping of highly (U87) vs. poorly (A549/8) angiogenic tumors provided results comparable to culture models. Therefore, FT-IR imaging allows detecting subtle chemical changes in tumors, which might be useful for diagnosis.
Abstract: The endoplasmic reticulum (ER) has emerged as a major site of cellular homeostasis regulation, particularly in the unfolded protein response, which is being found to play a major role in cancer and many other diseases. Here, we address ER-mediated signaling and regulations in the context of environmental challenges in cancer, such as hypoxia, angiogenesis, and chemotherapeutic resistance, and we discuss how ER-resident molecular machines become deregulated and involved in cancer-related pathology. Further exploration of how the ER senses, signals, and adapts to stress may redefine and deepen our understanding of its functions in cancer pathobiology.
Abstract: FT-IR spectrometry has proved to be a useful tool for determining a series of plasma molecular concentrations. Dedicated experiments were first performed to test the analytical performance that could be obtained by FT-IR spectrometry using a synthesized N3-peptide exhibiting a -N3 absorption centered at 2110 cm(-1), a spectral region where no organic material of biological samples absorbs. Further, we investigated whether this technology was able to allow quantification of metabolic parameters (glucose and lactic acid) within plasma, cells, and tissues as an alternative method to the "classical" biochemical approaches, which require sophisticated biological material treatment and expensive reagents. For this purpose we used a series of plasma samples to determine glucose and lactic acid concentrations, which are common markers of cancer growth. We compared the results of the main spectral data treatments commonly achieved for FT-IR data analysis, such as univariate (Beer-Lambert) or multivariate (PLS) calibrations, as well as the deconvolution of the spectral interval of interest (1200-900 cm(-1)). No significant differences were found regarding the analytical performances of these methods. Spectral deconvolution was finally undertaken on cultured and on xenografted cells (U87 glial cells implied in human gliomas) to determine glucose and lactic acid concentrations. In this case, qualification was allowed by FT-IR imaging on the cellular models since biochemical approaches are not efficient to reach metabolic concentrations at the cellular level while keeping tissue organization.
Abstract: Previously we have shown that hypoxia strongly induces the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) genes in several cancer cell lines via a HIF-dependent mechanism. In this paper we studied the expression and hypoxic regulation of PFKFB-4 and PFKFB-3 mRNA as well as its correlation with HIF-1alpha, HIF-2alpha, VEGF and Glut1 mRNA expression in the pancreatic cancer cell line Panc1 and two gastric cancer cell lines MKN45 and NUGC3. This study clearly demonstrated that PFKFB-3 and PFKFB-4 mRNA are expresses in MKN45, NUGC3 and Panc1 cancers cells and that both genes are responsive to hypoxia in vitro. However, their basal level of expression and hypoxia responsiveness vary in the different cells studied. Particularly, PFKFB-3 mRNA is highly expressed in MKN45 and NUGC3 cancer cells, with the highest response to hypoxia in the NUGC3 cell line. The PFKFB-4 mRNA has a variable low basal level of expression in both gastric and pancreatic cancer cell lines. However, the highest hypoxia response of PFKFB-4 mRNA is found in the pancreatic cancer cell line Panc1. The basal level of PFKFB-4 protein expression is the highest in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with the highest response to hypoxia in the pancreatic cancer cell line. Further studies showed that PFKFB-3 and PFKFB-4 gene expression was highly responsive to the hypoxia mimic dimethyloxalylglycine, a specific inhibitor of HIF-alpha hydroxylase enzymes, suggesting that the hypoxia responsiveness of PFKFB-3 and PFKFB-4 genes in these cell lines is regulated by the HIF transcription complex. The expression of VEGF and Glut1, which are known HIF-dependent genes, is also strongly induced under hypoxic conditions in gastric and pancreatic cancer cell lines. The levels of HIF-1alpha protein are increased in both gastric and pancreatic cancer cell lines under hypoxic conditions. However, the basal level of HIF-1alpha as well as HIF-2alpha mRNA expression and their hypoxia responsiveness are different in the MKN45 and NUGC3 cancer cells. Thus, the expression of HIF-1alpha mRNA is decreased in both gastric cancer cell lines treated by hypoxia or dimethyloxalylglycine, but HIF-2alpha mRNA expression is not changed significantly in NUGC3 and slightly increased in MKN45 cells. Expression of PFKFB-4 and PFKFB-3 was also studied in gastric cancers and corresponding nonmalignant tissue counterparts from the same patients on both the mRNA and protein levels. The expression of PFKFB-3 and PFKFB-4 mRNA as well as PFKFB-1 and PFKFB-2 mRNA was observed in normal human gastric tissue and was increased in malignant gastric tumors. The basal level of PFKFB-4 protein expression in gastric cancers was much higher as compared to the PFKFB-3 isoenzyme. In conclusion, this study provides evidence that PFKFB-4 and PFKFB-3 genes are also expressed in gastric and pancreatic cancer cells, they strongly respond to hypoxia via a HIF-1alpha dependent mechanism and, together with the expression of PFKFB-1 and PFKFB-2 genes, possibly have a significant role in the Warburg effect which is found in malignant cells.
Abstract: Angiogenesis in tumors is controlled by the so-called 'angiogenic switch' which allows the passage from low invasive and poorly vascularized tumors to highly invasive and angiogenic tumors. A number of cellular stress factors such as hypoxia, nutrient deprivation or inducers of reactive oxygen species (ROS) are important stimuli of angiogenic signalling. The HIF system plays a significant role in several of these effects and the molecular mechanisms of its regulation have recently been characterized. In addition, HIF-independent mechanisms have been described which involved number of other molecules and transcription factors such as nuclear factor-(kappa)B (NF-(kappa)B) and p53. p53 is an important intracellular mediator of the stress response and is now also recognized as a modifier of the angiogenic response. p53 may interact with the HIF system but may also have direct effects on angiogenesis regulators or interfere with translation mechanisms of angiogenesis factors.
Abstract: Angiogenesis is a key process in the pathogenesis of inflammatory arthritis. Angiogenin is one of the most potent inducers of neovascularization in experimental models in vivo. To look for evidence that angiogenin is involved in inflammatory joint disease, we examined plasma and synovial fluid (SF) samples from rheumatology patients and synovial fibroblast cell culture supernatants. Angiogenin levels were determined by radioimmunoassay and ELISA. Plasma angiogenin concentrations ranged from 96 to 478 ng/ml, with no significant difference between patients and normal controls. In SF, angiogenin concentrations were significantly higher in patients with acute or chronic synovitis (rheumatoid arthritis (RA): median, 104 ng/ml; range 13-748, n = 14; crystal-induced arthritis (CIA): median, 149 ng/ml; range, 37-616, n = 14, and other chronic inflammatory arthritis: median, 42 ng/ml; range, 15-205; n = 9) than in the 18 patients with osteoarthritis (OA) (median, 20 ng/ml; range 8-116) (P < 0.0001, anova). Angiogenin levels in SF from RA patients in remission with secondary OA were similar to those achieved in primary OA, and decreased in parallel with the resolution of acute gout. Angiogenin protein was released by cultured synovial fibroblasts from OA and RA patients, and reached 1.18 ng/106 cells/day. These data suggest that angiogenin may mediate local inflammation in arthritis via effects on angiogenesis and leucocyte regulation.
Abstract: Pyrimidine-specific ribonucleases are a superfamily of structurally related enzymes with distinct catalytic and biological properties. We used a combination of enzymatic and non-enzymatic assays to investigate the release of such enzymes by isolated cells in serum-free and serum-containing media. We found that human endothelial cells typically expressed large amounts of a pancreatic-type RNase that is related to, if not identical to, human pancreatic RNase. This enzyme exhibits pyrimidine-specific catalytic activity, with a marked preference for poly(C) substrate over poly(U) substrate. It was potently inhibited by placental RNase inhibitor, the selective pancreatic-type RNase inhibitor Inhibit-Ace, and a polyclonal antibody against human pancreatic RNase. The enzyme isolated from medium conditioned by immortalized umbilical vein endothelial cells (EA.hy926) possesses an amino-terminal sequence identical to that of pancreatic RNase, and shows molecular heterogeneity (molecular weights 18,000-26,000) due to different degrees of N-glycosylation. Endothelial cells from arteries, veins, and capillaries secreted up to 100 ng of this RNase daily per million cells, whereas levels were low or undetectable in media conditioned by other cell types examined. The corresponding messenger RNA was detected by RT-PCR in most cell types tested so far, and level of its expression was in keeping with the amounts of protein. The selective strong release of pancreatic-type RNase by endothelial cells suggests that it is endowed with non-digestive functions and involved in vascular homeostasis.
Abstract: A radio-ribonuclease inhibitor assay based on the interaction of 125I-angiogenin with ribonuclease inhibitor (RI) was used to detect pancreatic-type ribonucleases and potential modulators of their action. We show that highly basic proteins including the homopolypeptides poly-arginine, poly-lysine and poly-ornithine, core histones, spermatid-specific S1 protein and the protamines HP3 and Z3 were strong inhibitors of angiogenin binding to RI. A minimum size of poly-arginine and poly-lysine was required for efficient inhibition. The inhibition likely resulted from direct association of the basic proteins with the acidic inhibitor, as RI bound to poly-lysine and protamines while 125I-angiogenin did not. Antagonists of the angiogenin-RI interaction are potential regulators of either angiogenin-triggered angiogenesis and/or intracellular RI function, depending on their preferential target.
Abstract: Significant amounts of ribonuclease inhibitor protein are present in human and rat erythrocytes, cells that are essentially devoid of ribonuclease or functional RNA. The protein from human erythrocytes is indistinguishable from human placental ribonuclease inhibitor protein by immunological and biochemical criteria. Each inhibitor forms an equimolar complex with bovine pancreatic ribonuclease A and is inactivated by treatment with the sulfhydryl reagent p-(hydroxymercuri)benzoate. Amino acid composition and several cycles of amino acid sequence analysis also showed apparent identify of the erythrocyte and placental proteins. We calculate a level of 1.5-3.5 x 10(4) molecules of active inhibitor per erythrocyte, most or all of which occurs in an uncomplexed form since inactivation of the inhibitor revealed barely detectable levels of RNase activity. Immunogold localization showed a high level of labeling and a uniform distribution of gold particles in the cytoplasm of erythrocytes, while little inhibitor activity was found in association with isolated red blood cell membranes. Oxidative stress on isolated red cells resulted in a decrease in the level of reduced glutathione and a gradual and irreversible loss of inhibitor activity; inhibitor disappeared from the cytosol and became associated with nascent Heinz bodies. We suggest a role for this protein in the metabolism and aging process of the erythrocyte.
Abstract: The requirement of serum in cell culture is a major limitation for studies on secreted ribonucleases (RNases) because serum contains a high amount of ribonucleolytic activity. Defined culture condition is thus of interest to improve our knowledge of the RNase biology. We report here that cells from three different types and origins, Chinese hamster lung fibroblasts, bovine smooth muscle cells, and human endothelium-derived EA.hy926 cells, proliferate consistently in the presence of a basal medium supplemented with bovine serum albumin, high-density lipoproteins, basic fibroblast growth factor, insulin, and transferrin. Using a new quantitative radio-RNase inhibitor assay, two distinct ribonucleolytic assays, and a radioimmunoassay against angiogenin, it is shown that RNases became apparent in media conditioned by cell monolayers. Both the hamster lung fibroblast and the EA.hy926 cell lines secreted larger amounts of RNase inhibitor-interacting factors and RNase activity than normal smooth muscle cells. The serum-free medium represents an alternative way to grow these cells and allows investigation of biosynthesis and functions of RNases in culture. It should be useful to identify and quantitate unambiguously specific members of the RNase family secreted by normal versus tumor cells in culture.
Abstract: Angiogenin is a secreted polypeptide that induces neovascularization in vivo. The expression of angiogenin by human cells in culture was investigated by using a specific radioimmunoassay and by cDNA hybridization. Angiogenin immunoreactivity was widely but differentially produced by anchorage-dependent growing cells including vascular endothelial cells from saphenous and umbilical veins, aortic smooth muscle cells, fibroblasts (from embryos, new-borns and adults), and tumour cells. Endothelial cells from saphenous veins and the endothelium-derived EA.hy926 cell line released immunoreactivity whatever the stage of the culture, including release at the lag phase, during exponential growth and at the confluent phase. However, the rate of accumulation of angiogenin varied as a function of EA.hy926 cell density. As compared to anchored cells, normal peripheral blood cells and tumour cells of myelomonocytic and megakaryocytic origin did not noticeably secrete angiogenin except at low levels. A myeloma cell line supernatant contained as much angiogenin cross-reactivity as did anchored cells, while four tumour T-cell lines expressed the cross-reactivity at different levels, i.e. from undetectable levels to a high level. A 0.9-kb angiogenin messenger RNA was detected by Northern-blot analyses in a variety of representative cells correlating with the presence of immunoreactivity in the cell-culture media. The widespread expression pattern of angiogenin suggests a physiological function that is not restricted to the neovascularization process.
Abstract: The rabbit corneal angiogenesis assay was modified to allow quantification of the neovascular response induced by growth factors. Basic Fibroblast Growth Factor (bFGF) was used for this experiment. bFGF was diluted in phosphate buffer solution (PBS) in such a way that 2 microliter of solution contained concentrations ranging from 17 to 1,200 ng. Each drop was absorbed by a 1 x 2 mm particle of a dried 70% hydratable hydrogel and implanted into a mid-stroma corneal pocket, 2 mm from the limbus. Iodinated bFGF was used to measure growth factor diffusion in the cornea, which was found to be isotropic. Autoradiography showed that bFGF was stored in the cornea at both epithelial and endothelial level. Corneal neovascularization occurred on the second day after implantation and was maximal on the 7th day. At this time the neovascular surface was measured by planimetry on corneal photographs and compared with controls. This method allows precise definition of the dose of any substance to be implanted into the cornea and induces a rapid neovascular response, thus allowing quantitative evaluation of neovascularization within one week. A neovascular response was detectable for doses as low as 35 ng of bFGF and increased proportionally to the dose of bFGF implanted.
Abstract: Subconfluent Chinese hamster lung fibroblast cells (CCL39) which express high- and low-affinity binding sites for basic fibroblast growth factor (bFGF) were used to study bFGF internalization. Kinetics at 37 degrees C indicated that this process was complex and involved various pathways with regard to the ligand concentration used. Internalization with 6 to 45 pM of 125I-r-bFGF led to a steady state that lasted up to 3 h without any appearance of 125I-labeled degradation products in the cell-culture medium, suggesting that the endocytosis reached equilibrium. Furthermore, binding data at steady state, at 37 degrees C, revealed a two-phase Scatchard curve suggesting the involvement of two families of interaction sites in the process of internalization. Apparent dissociation constants were estimated to be 20 pM and 58 nM, respectively, and the number of bFGF molecules involved per cell, 4300 and 1.3 x 10(6), respectively. These data were in good agreement with those obtained from binding experiments at equilibrium at 4 degrees C. Besides, higher concentrations of 125I-r-bFGF (greater than 47 pM) induced an internalization process which did not reach steady state and was not saturable. These results suggest that CCL39 cells could internalize bFGF by various pathways involving high- and low-affinity binding sites.
Abstract: Using either acidic (pH 2.5) or trypsic treatments, we demonstrated that 125I-labeled basic Fibroblast Growth Factor (125I-bFGF) was submitted to an internalization process on responsive Chinese hamster lung fibroblasts (CCL39) at 37 degrees C. Various experiments based on the measurement of cell-associated radioactivity, as well as on research of degradated products of 125I-bFGF in cellular supernatants, showed that most of the internalized radioactivity remained intracellularly located after up to 5 hr of incubation. Analyses of this radioactivity by NaDodSO4-PAGE revealed the presence of labeled peptides issued from the limited processing of the native 125I-bFGF form (17 kD) and whose molecular weights were estimated to be 9 and 6 kD. Kinetic experiments indicated that proteolysis of the 125I-bFGF began early on incubation (less than 30 min) and led to a prolonged preservation of the 9- and 6-kD peptides which were still detectable after 13 hr of incubation. Preincubation of the cells with different lysosomotropic agents completely inhibited the proteolysis, indicating that this event occurred probably in an intracellular acidic compartment. Two enzyme inhibitors, leupeptin and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), were also shown to interfere with the formation of both 9- and 6-kD peptides, thus suggesting a way to control the appearance of these fragments, and hence to determine their potential intracellular role.
Abstract: In a previous work, we showed and compared the wound healing properties of aFGF and bFGF topically administered on totally de-epithelialized rabbit corneas. Pharmacokinetic and autoradiographic studies were then performed to investigate the sites of accumulation of bFGF in ocular structures, both on de-epithelialized and intact rabbit eyes. After one single instillation of 125I-bFGF, all the ocular structures were dissected and the measurement of the radioactivity allowed to establish kinetic curves. The results showed a very important and early fixation of bFGF on denuded cornea (10 minutes) and a posterior distribution of the drug between 10 and 30 minutes. A second accumulation of bFGF in the anterior segment appeared 8 hours after application and then decreased till the 48th hour. These findings were confirmed by the macroautoradiographies and the microautoradiographies pointed out the fixation of bFGF not only at the location of the Bowman membrane, but also on the corneal endothelium. These experiments also demonstrated the systemic diffusion of bFGF into the untreated controlateral eye. The integrity of bFGF in the cornea and other structures was then confirmed by SDS PAGE followed by autoradiography. In the intact eye, bFGF was shown to penetrate in extremely low amounts, illustrating the major role of the corneal barrier. For a therapeutic use bFGF may be recommended as an efficient wound healing agent for epithelial but also endothelial defects. Its eventual unwanted side effects must be kept in mind to perfect an efficient low dose and short term clinical treatment.
Abstract: Two bovine brain-derived growth factors, BDGF I and BDGF II, were isolated using the same extraction procedure as previously described for eye-derived growth factors (EDGF). The hypothesis that these growth factors were identical to EDGF I and EDGF II, respectively, was supported by their similar molecular weights (16,000 and 15,000, respectively) and isoelectric points (9.0 and 5.0, respectively), their identical retention behavior on reverse-phase chromatography and their similar amino acid compositions. From studies on their binding properties to cell surfaces, competition between EDGF I and BDGF I as well as competition between EDGF II and BDGF II to the same receptor was observed. The amino terminal sequence of EDGF II (1-16) was shown to be identical to the amino acid residues (7-22) of the acidic FGF, strongly confirming our observations on the identity of the factors isolated from bovine brain and retina.
Abstract: The labeling pattern of mouse embryonic eye frozen sections incubated with radioiodinated brain acidic and basic fibroblasts growth factors (aFGF and bFGF) was investigated by autoradiography. Both growth factors bind to basement membranes in a dose-dependent way, with a higher affinity for bFGF. Similar data were obtained with eye-derived growth factors (EDGF), the retinal forms of FGF. There was a heterogeneity in the affinity of the various basement membranes toward these growth factors. The inner limiting membrane of the retina and the posterior part of the lens capsule have a higher binding capacity than the posterior part of the Bruch's membrane. The specificity of the growth factor-basement membrane interaction was demonstrated by the following experiments: (i) an excess of unlabeled growth factor displaced the labeling; (ii) unrelated proteins with different isoelectric points--gelatin, serum albumin, histones--did not modify the labeling; and (iii) iodinated EGF or PDGF did not label basement membrane. In order to get a better understanding of the nature of this binding, we performed the incubation of the frozen sections with iodinated FGFs preincubated with various compounds: (i) heparin which is known to have a strong affinity for aFGF and bFGF partially decreases the labeling, and (ii) chondroitin sulfate B and dextran sulfate at high concentrations were also partially effective. In addition, enzymatic treatment of the sections reveals that only heparitinase, not collagenase or chondroitinase ABC, completely prevents the labeling without destroying the overall structure of the basement membrane. An antibody against the proteic part of EHS mouse proteoheparan sulfate does not affect the signal. Esterification of the acidic groups cancelled the binding. These results demonstrate that FGFs bind specifically to basement membranes, probably on the polysaccharidic part of the proteoheparan sulfate, and suggest that this type of interaction may be a general feature of the mechanism of action of these growth factors.
Abstract: Basic or acidic forms of FGF, a potent mitogen for Bovine Epithelial Lens cells caused a rapid and transient rise in cytoplasmic Ca2+ followed by an increase in intracellular pH of 0.4 units. When cells were labeled at equilibrium with [3H]-inositol, no significant breakdown of polyphosphoinositides (in the presence of 20 mM LiCl) could be detected in response to 10-100 ng/ml of FGF. Similarly, fetal calf serum efficiently reinitiated DNA synthesis in these cells with little stimulation of polyphosphoinositide hydrolysis. In contrast, prostaglandin F2 alpha and angiotensin II, two weak mitogens for BEL cells, were found potent agonists of polyphosphoinositide breakdown. These results strongly indicate that the mitogenic action of FGF is not coupled to phospholipase C activation, a conclusion consistent with the fact that the FGF-induced [Ca2+]i rise is strictly dependent upon external Ca2+.
Abstract: Eye-derived growth factor I (EDGF-I), the retinal form of the basic fibroblast growth factor, has been purified to homogeneity from bovine retina by heparin-Sepharose chromatography. The radioiodinated EDGF-I retained full mitogenic activity and was used to study the interaction of the growth factor with bovine epithelial lens cells. We showed that 125I-labeled EDGF-I bound in a saturable and reversible manner to a specific cellular receptor. Scatchard analysis of the equilibrium binding gave a Kd of 53 X 10(-12) M with approximately equal to 20,000 binding sites per cell. Crosslinking experiments using two homobifunctional reagents induced the formation of a specific major complex with a Mr of approximately equal to 145,000, as determined by NaDodSO4/PAGE, and independent of reducing conditions. These data establish the existence of a receptor for the basic growth factor derived from neural tissues and give an estimation of the size of this receptor at Mr 130,000.
Abstract: Eye Derived Growth Factor (EDGF) is the genus name for growth factor activities found in several ocular tissues. Purification from bovine retina by Cibacron blue affinity chromatography has previously given a fraction which can induce target cell proliferation at doses of 30 ng per ml of culture medium. Radioimmunoassay using a labelled synthetic decapeptide [Tyr 10]--FGF (1-10) including the 9 N terminal amino acids of brain Fibroblast Growth Factor (FGF) indicated that EDGF contained a FGF-like material. Further purification of Cibacron blue purified EDGF with heparin sepharose chromatography yielded two active fractions after elution with a sodium chloride gradient. One fraction named EDGF I eluted between 1.3 and 1.5 M NaCl and accounted for over 50% of the input biological activity and comigrated with purified FGF on SDS PAGE at a molecular weight of 16,000 d as a single band. FGF competed with EDGF I for binding to specific receptors on bovine epithelial lens cells. We conclude that retina contains a growth factor activity (EDGF I) similar if not identical to FGF.
Abstract: Basic or acidic fibroblast growth factor (FGF), alone, was found to be as potent as alpha-thrombin to reinitiate DNA synthesis in G0-arrested Chinese hamster lung fibroblasts (CCL39). Basic FGF at 50 ng/ml or thrombin at 1 unit/ml rapidly initiated early events such as cytoplasmic alkalinization (0.2-0.3 pH units), rise in cytoplasmic Ca2+, phosphorylation of ribosomal protein S6 and increased c-myc expression, followed by a 30-40-fold increase in labeled nuclei. Whereas thrombin is a potent activator of phospholipase C as judged by the rapid release of inositol trisphosphate, inositol bisphosphate and by the massive accumulation of total inositol phosphate (IP) in the presence of 20 mM Li+, FGF failed to induce the breakdown of polyphosphoinositides in quiescent CCL39 cells. Indeed, no inositol trisphosphate nor inositol bisphosphate could be detected in response to FGF; in presence of Li+ the total IP release never exceeded 8% of the IP released by the action of thrombin. Two additional findings indicated that FGF and thrombin activate different signaling pathways. First, we found that, in contrast to thrombin, the FGF-induced rise in the cytoplasmic free Ca2+ concentration measured by quin-2 fluorescence, is strictly dependent upon the presence of Ca2+ in the external medium. Second, we found that FGF failed to activate protein kinase C as judged by the epidermal growth factor-receptor binding assay. Treatment of the cells with either thrombin or phorbol esters, rapidly inhibited 125I-labeled epidermal growth factor binding (50-60%). Basic or acidic FGF had no effect. We conclude that: the FGF-receptor signaling pathway is not coupled to phospholipase C activation, and early mitogenic events and reinitiation of DNA synthesis can be initiated independently of inositol lipid breakdown and protein kinase C activation.
Abstract: Several ocular tissues have been shown to contain growth factor activity designated under a generic name as Eye Derived Growth Factor. Purification from bovine retina was undertaken and a fraction which could induce target cells to proliferate at doses of 5 ng per ml of culture medium was obtained. Using heparin sepharose chromatography we now show that this mitogenic activity can be fractionated into three different activities. Crude extract of bovine retina used as starting material was separated into two major fractions, one with no affinity for heparin and which was named Eye Derived Growth Factor III, and one with a strong affinity for heparin and eluted from the column with 1.4 M NaCl named Eye Derived Growth Factor I. This fraction EDGF I induces cell proliferation at doses of 100 pg/ml of culture medium. A 10(5) fold purification was achieved by this single chromatography step. Cibacron Blue purified EDGF was also further fractionated by heparin sepharose. All biological activity was found to bind to heparin. One fraction eluted at 1 M NaCl named Eye Derived Growth Factor II had a biological activity at doses of 1 ng while the other growth factor was the EDGF I with biological activity at 25 pg. At this step of purification EDGF I runs as a single band on SDS polyacrylamide gel at a molecular weight of 17 000 d. These data strongly suggest that Eye Derived Growth Factors I and II are respectively similar to Brain Fibroblast Growth Factor and to Endothelial Cell Growth Factor from hypothalamus.
Abstract: A series of derivatives of estrone with fluorescent dyes (dansyl or coumarin) coupled at different positions on the steroid molecule, have been synthesized. These derivatives were tested for their quantum yields and their binding properties were determined with respect to rat alpha 1 fetoprotein. Derivatives at C-3 (estrone-3-hemisuccinate-dansyl-cadaverine and estrone-3-dansyl) compete with estrone for binding to the fetal protein; however derivates of estrone at C-6 (estrone-6-carboxymethyloxime-dansyl-cadaverine) and at C-17 (estrone-17-carboxymethyloxime-coumarine, estrone-17-carboxymethyloxime-dansyl-cadaverine and estrone-17-dansyl-hydrazine) compete poorly or not at all. The association constant of the radioactive derivative estrone-3-dansyl [3H] with rat alpha 1 fetoprotein was measured directly: the same number of high affinity binding sites (0.6) as that for estrone was found with an apparent association constant of 3.7 X 10(6) M-1. In addition to the high affinity binding sites, a low affinity class of binding sites was found which corresponds to the binding of the dansyl fraction of the fluorescent steroid derivative.
