Relationship between serum concentrations of the growth factor pleiotrophin and pleiotrophin-positive tumors.

BACKGROUND: Growth factors produced by tumor cells are essential for tumor expansion and may be useful in monitoring tumor progression or therapeutic efficacy if the factors are released into the circulation. In this study, we measured serum levels of pleiotrophin, a secreted heparin-binding growth and angiogenesis factor, in mice bearing human tumor xenografts to determine whether these levels reflected overall tumor burden, and we examined the relationship between tumor expression of pleiotrophin and serum levels of this factor in patients with cancer. METHODS: Pleiotrophin in serum from mice and humans was measured by use of a highly sensitive enzyme-linked immunosorbent assay. For the clinical studies, serum specimens were obtained from 193 patients with various cancers of the gastrointestinal tract and from 28 healthy control subjects. In a subset of 64 cancer patients, serum levels of pleiotrophin were measured at the time of surgery, and tumor expression of this factor was detected immunohistochemically. All P values are two-sided. RESULTS: In mice, serum pleiotrophin levels were found to increase as a function of tumor size. In humans, elevated serum pleiotrophin levels were found in patients with pancreatic cancer (n = 41; P<.0001) and colon cancer (n = 65; P = .0079) but not in patients with stomach cancer (n = 87; P =.42). A statistically significant positive association was found between elevated levels of pleiotrophin in serum drawn at the time of surgery and expression of this factor by tumors (P<.0001). In both mice and humans, serum pleiotrophin levels dropped after successful tumor removal. CONCLUSIONS: Elevated serum pleiotrophin levels can indicate the presence of tumors expressing this factor. Monitoring serum levels of pleiotrophin may prove useful in determining the pharmacologic efficacy of cytotoxic or anti-pleiotrophin therapy.

Epitopes of the 44-68 human parathyroid hormone fragments: the importance of specific hydrophilic peptide sequences.

Several pentapeptides included in the 44-68 sequence of human parathyroid hormone (hPTH) were synthesized simultaneously on benzhydrylamine and m-nitrobenzhydrylamine resins. The first polymer gave the free peptide and the second the peptidyl-resin complex. An ELISA test carried out with each peptidyl-resin complex showed that all the anti-44-68 hPTH antibodies raised in different animal species are directed against the same hPTH pentapeptidic sequence. This sequence is very hydrophilic and is specific to the hormone. This study demonstrates the importance of specific peptide chains in an epitope.

Immunolocalization of acidic and basic fibroblast growth factors during mouse odontogenesis.

Acidic and basic fibroblast growth factors (aFGF and bFGF), are both known to bind to extracellular matrix components, particularly proteoheparin sulfates, and to regulate in vitro proliferation, differentiation and morphology of cells of neuroectodermal and mesodermal origins. Their patterns of distribution were studied during mouse odontogenesis by means of indirect immunofluorescence and immunoperoxidase histochemistry on frozen fixed sections and after Bouin's fixative and paraffin embedding. Localization of aFGF on frozen fixed sections was observed in the oral epithelium, dental lamina and oral mesenchyme (day-12 of gestation), the stellate reticulum and oral epithelium (day-14), the stratum intermedium and at the basal and apical poles of preameloblasts at bell stage. After birth aFGF epitopes were localized within the predentin-dentin area, the stratum intermedium and at the secretory pole of ameloblasts. There was no staining with anti-aFGF antibodies after Bouin's fixative and paraffin embedding. In contrast, using this protocol, intense stainings were found with anti-bFGF antibodies predominantly within dental and peridental basement membranes and mesenchyme: staining of the dental basement membranes was transient (bud and cap stage) and discontinuous; a preferential concentration of bFGF epitopes in the condensed dental mesenchyme of incisors (cap stage) and the dental papillae mesenchymal cells of molars (bell stage) was observed in the posterior and the cervical part of tooth germs. An intense immunostaining of the stellate reticulum with anti-bFGF antibodies was also found on paraffin sections from bud to bell stage.(ABSTRACT TRUNCATED AT 250 WORDS)

HB-GAM/pleiotrophin: localization of mRNA and protein in the chicken developing leg.

The heparin-binding growth-associated molecule HB-GAM (also named pleiotrophin) is a developmentally-regulated protein that belongs to a new family of heparin-binding molecules with putative functions during cell growth and differentiation. In order to study the localization of HB-GAM during chicken embryogenesis, we produced specific monoclonal antibodies to this factor. HB-GAM protein is first observed at stage 23 in the developing nervous system and later in the forming cartilage. We present an investigation of the HB-GAM mRNA expression and HB-GAM protein distribution in the developing leg by in situ hybridization and immunocytochemical studies. We focused our attention on the development of the tibia, where the HB-GAM protein appears at stage 27-28, i.e., just after the condensation of the mesodermal precursor cells of the chondrocytes. The protein then progressively accumulates in the central part of the embryonic cartilage (diaphysis). It persists until stage 42-44 in the regions where hypertrophic cartilage is being replaced by bone marrow. In contrast to the protein, the transcript is first detected at stage 26-27 and later expressed essentially in the epiphysis until stage 37. Therefore the localization of the mRNA does not parallel that of the protein and our data suggest a long half-life of the protein in the hypertrophic cartilage. In addition, the layer of stacked cells surrounding the cartilage core (usually considered as the osteoprogenitor cells) clearly expresses the HB-GAM message between stages 30-37 whereas differentiated osteoblasts do not. Furthermore, the distribution of HB-GAM protein in the osteoblast/osteoid layer suggests an involvement of this protein in early steps of osteogenesis. HB-GAM is absent from the newly formed bone.

Acidic fibroblast growth factor (aFGF)-like immunoreactivity in the optic nerve.

Acidic fibroblast growth factor (aFGF)-like immunoreactivity was examined in the optic nerves of 1- to 25-month-old Wistar rats, 0.5- to 7-year-old bovine animals and normal human adults (24 and 35 years old), using cryostat sections incubated with a rabbit polyclonal antibody specific for aFGF. The immunoreactivity was associated with glial cells, and was localized predominantly in the nucleus. The presence of endogenous aFGF in the optic nerve of adult subjects and 'old' rats suggests that aFGF could play a role in the survival of retinal ganglion cells and their axons during aging.

Localization of acidic fibroblast growth factor in proliferative vitreoretinopathy membranes.

The pathogenesis of proliferative vitreoretinopathy (PVR) membranes remains poorly understood. We have studied the presence of acidic fibroblast growth factor (aFGF), a potent mitogen for many cells, within these membranes. We have used affinity purified monospecific anti-aFGF polyclonal antibodies, in conjunction with highly sensitive immunofluorescence techniques. The labelling was exclusively localized to cell bodies and was absent from the extracellular matrix. Double labelling techniques revealed that all cytokeratin positive cells (probably pigmented epithelial cells) and macrophages contained aFGF-like immunoreactivity, whilst glial cells were unlabelled. Appropriate controls indicated the specificity of the antibodies. Hence, the presence of this mitogenic molecule within certain cell types constituting PVR membranes may contribute to the pathogenesis.

Fibroblast growth factor phosphorylation and receptors in rod outer segments.

Acidic and basic fibroblast growth factors (aFGF and bFGF) have been isolated and purified from rod outer segments (ROS). aFGF is tightly bound to ROS membranes and can be specifically released by ATP. We show that this mechanism is dependent on the phosphorylation of aFGF itself. Phorbol 12-myristate 13-acetate (PMA) enhances this phenomenon independently of rhodopsin phosphorylation. This demonstrates that aFGF release from ROS membranes is dependent on its phosphorylation by endogenous kinase C. In addition specific binding sites for exogenous FGFs have been identified on ROS and disc membranes. A single high affinity site with a Kd of 40 pM was present in intact ROS while an additional low affinity site with a Kd of 300-600 pM was present in leaky ROS or in disc membranes. Light or ATP modified neither these Kd nor the apparent number of sites. The presence of specific receptors for FGFs and the kinase C dependent release of endogenous membrane bound aFGF suggest an autocrine mechanism which may be involved in photoreceptor cell biology.

Acidic fibroblast growth factor (aFGF) in developing normal and dystrophic (mdx) mouse muscles. Distribution in degenerating and regenerating mdx myofibres.

Affinity purified polyclonal antibodies directed against human recombinant acidic FGF (aFGF), were used in immunofluorescence studies to localize this growth factor in several normal and dystrophic (mdx) mouse skeletal muscles. The expression of aFGF was detected throughout the life of both the control and mdx mice. In striated muscles, examined up to 3 weeks postnatal, aFGF was localized around the myofibres and this pattern was consistent in both mdx and the normal counterpart strain. However, the intensity of the signal was much stronger in the mdx strain. In mdx mouse skeletal muscles, examined during the acute phase of degeneration and regeneration (3-14 weeks) aFGF was localized around the myofibres, in approximately 60% of the nuclei of newly formed or regenerated myofibres and also in the pockets of necrosis which represented actively degenerating myofibres. In normal mouse skeletal muscles, studied over the same period, the antibodies localized aFGF mainly to the periphery of the muscle fibres. The augmentation of aFGF observed by immunofluorescence in mdx mouse muscles was confirmed by enzyme immunoassay (EIA) analysis of the same muscles over the same period of time. The data from the EIA indicated a 3.5-fold increase in aFGF in mdx as compared to normal muscles at 3 weeks, and an approximate 26-fold increase during the period of active degeneration-regeneration. This increased concentration of aFGF noted in the mdx muscles suggests that this endogenous aFGF may participate in the high level of regenerative activity observed in mdx mouse.

Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways.

Pleiotrophin (PTN) is a developmentally regulated protein that has been shown to be involved in tumor growth and metastasis presumably by activating tumor angiogenesis. To clarify the potential angiogenic activity of PTN and to analyze the signaling pathways involved in this process, we used an in vitro model of Human Umbilical Vein Endothelial Cells (HUVEC). We show that PTN was mitogenic toward a variety of endothelial cells including HUVEC, stimulated HUVEC migration across a reconstituted basement membrane and induced the formation of capillary-like structures by HUVEC grown as 3D-cultures in Matrigel or collagen. The signaling pathways triggered following endothelial cell stimulation by PTN were studied by using pharmacological inhibitors of the Phosphoinositide-3 kinase (PI3K) and endothelial Nitric Oxide Synthase (eNOS), two enzymes that have been shown to be crucial in the angiogenic response to Vascular Endothelial Growth Factor (VEGF). Whereas wortmannin (a PI3K inhibitor) and L-NAME (an eNOS inhibitor) dramatically reduced HUVEC growth induced by VEGF, only the former inhibitor reduced the growth induced by PTN and to a lesser extent that stimulated by basic Fibroblast Growth Factor. Thus, our results indicate that PTN induces angiogenesis and utilizes PI3K- but not eNOS-dependent pathways for its angiogenic activity.

Activation of anaplastic lymphoma kinase receptor tyrosine kinase induces neuronal differentiation through the mitogen-activated protein kinase pathway.

Anaplastic lymphoma kinase (ALK) is a novel neuronal orphan receptor tyrosine kinase that is essentially and transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. To determine whether ALK could play a role in neuronal differentiation, we established a model system that allowed us to mimic the normal activation of this receptor. We expressed, in PC12 cells, a chimeric protein in which the extracellular domain of the receptor was replaced by the mouse IgG 2b Fc domain. The Fc domain induced the dimerization and oligomerization of the chimeric protein leading to receptor phosphorylation and activation, thus mimicking the effect of ligand binding, whereas the wild type ALK remained as a monomeric nonphosphorylated protein. Expression of the chimera, but not that of the wild type ALK or of a kinase inactive form of the chimera, induced the differentiation of PC12 cells. Analysis of the signaling pathways involved in this process pointed to an essential role of the mitogen-activated protein kinase cascade. These results are consistent with a role for ALK in neuronal differentiation.

Heparin-binding sites of rat rhabdomyosarcoma cells with low and high metastatic capacity.

Proteins able to bind the iduronate containing glycosaminoglycans: heparin, heparan sulfate and dermatan sulfate, were detected in strongly (RMS 0) and weakly (RMS 8) metastatic rat rhabdomyosarcoma cell lines. The 35S-methionine-labeled proteins solubilized from the cell membranes were chromatographed on Heparin-Ultrogel affinity column. The main retained protein migrated with an apparent molecular size of 19 kDa on polyacrylamide gel electrophoresis from both cell lines. The 19 kDa protein exhibited a higher affinity for iduronate containing glycosaminoglycans than for the glucuronate containing chondroitin sulfates. It was immunologically distinct from acid and basic fibroblast growth factors. The membranes of the RMS 8 cells contained about a two times higher amount of labeled 19 kDa protein than the membranes of the RMS 0 cells. The decreased amount of the labeled heparin-binding proteins in the highly metastatic cell line is in agreement with the previously evidenced decreased receptor-mediated binding of the iduronate containing glycosaminoglycans by these cells.

Characterization of acidic and basic fibroblast growth factors in brain, retina and vitreous chick embryo.

We have purified acidic and basic fibroblast growth factors (c-aFGF, c-bFGF) from 11 day-old chick embryo brain, retina and vitreous by heparin-Sepharose chromatography and reverse phase HPLC. The analysis of their biological activity as well as their molecular weight indicates that they were analogous to basic or acidic human and bovine FGF. The ratio of c-aFGF to c-bFGF activity depended of the tissue. In brain c-aFGF represented 66% of the total mitogenic activity retained on the heparin-sepharose column and c-bFGF 34% while retina contained 16% of c-aFGF and 84% of c-bFGF; vitreous 78% of c-aFGF and 22% of c-bFGF. Like human aFGF, Heparin stimulated purified c-aFGF mitogenic activity in the absence of serum but inhibited the activity of the retina acid soluble extract, in the presence of foetal calf serum (FCS). Thus, chick embryo and adult human acidic and basic FGF respectively share the same biochemical properties. Since there are no blood vessels in chick retina or vitreous, their presence in these tissues suggests that angiogenesis is not the only role of these growth factors.

Retinoic acid-induced heparin binding protein (RIHB) binds to embryonal chondrocytes and cartilage primarily via proteoglycans.

Retinoic acid-induced heparin binding protein (RIHB) is a highly basic, secreted polypeptide expressed during early chick embryogenesis. We have characterized the binding of 125I-labeled RIHB to embryonal chondrocytes in culture. No saturable, high-affinity binding can be observed on these cells. Furthermore, no 125I-labeled RIHB was internalized into the chondrocytes at 37 degrees C. The low-affinity binding of 125I-labeled RIHB observed can be competed with another heparin binding factor, fibroblast growth factor 2, as efficiently as with unlabeled RIHB. The binding can also be almost completely inhibited by preincubation of the 125I-labeled RIHB with heparin or with a monoclonal antibody which recognizes the heparin binding site of both RIHB and HBNF. When cross-linking experiments are performed with 125I-labeled RIHB, specific RIHB-containing high-molecular-weight complexes are observed; however, these represent only a very small fraction of the bound material. Immunohistochemical analyses of embryonic wing cartilage demonstrate that a significant fraction of bound RIHB can be removed from unfixed tissue simply by rinsing with phosphate-buffered saline. The remaining RIHB can be removed partially by incubation with heparitinase I or III and completely when the incubation is performed with chondoitinase A, B, C. These results demonstrate that RIHB binds to embryonal chondrocytes and cartilage primarily through proteoglycans of both heparan sulfate and chondroitin sulfate types.

A new heparin binding protein regulated by retinoic acid from chick embryo.

A 19 KDa heparin binding protein was previously purified from chicken embryos. Essentially localized within basement membranes in early embryonic tissues, this protein is very rich in basic and cystein residues. Its N-terminal fragment is similar to corresponding fragment of two other proteins expressed during embryogenesis and postnatal period. Its synthesis and secretion are induced by retinoic acid in chicken myoblasts and fibroblasts. This new retinoic acid induced heparin binding protein (RI-HB) does stimulate neurite outgrowth and proliferation on PC12 cells. These results suggest that retinoic acid could regulate some aspect of differentiation and development by inducing the synthesis of a new family of growth and neurotrophic factors.

Retinoic acid-induced heparin-binding factor (RIHB) mRNA and protein are strongly induced in chick embryo chondrocytes treated with retinoic acid.

Retinoic acid-induced heparin-binding factor (RIHB) is a highly basic polypeptide expressed during early chick embryogenesis. We have examined the induction of RIHB by retinoic acid in chondrocytes isolated from the sterna of Day 15 chick embryos and the effects of exogenous RIHB on these cells. There is an induction of RIHB mRNA in chondrocytes which is dose dependent, with maximal levels of expression observed with concentrations of retinoic acid in the 10(-6) M range. RIHB mRNA is first observed 16 h after commencement of treatment, is maximal after 24-48 h, and is completely attenuated after 5 days. This transient pattern of expression is very similar to that of type X collagen; however, RIHB induction precedes that of type X collagen by about 24 h. The expression of both RIHB and type X collagen precedes the drop in keratan sulfate:chondroitin sulfate proteoglycan and type II collagen expression and the surge of fibronectin expression. The induction of RIHB mRNA is accompanied by an increased synthesis of the protein. No RIHB can be detected in untreated chondrocytes; however, large amounts are produced by cells treated with 5 x 10(-7) M retinoic acid. The protein is recovered mainly in the culture medium and bound to the extracellular matrix. Only a small amount can be detected in cell extracts. RIHB can be detected in the culture medium after 16-24 h and, unlike the mRNA, persists over the 5-day period examined. The effect of exogenous RIHB (purified from chick embryos) on chondrocyte proliferation and morphology was examined. When added to the culture medium in concentrations of up to 500 ng/ml RIHB had no effect on [3H]thymidine incorporation or cell morphology. Thus, RIHB is not the direct mediator of retinoic acid for these cells, but is strongly induced during the treatment.

Retinoic acid induced heparin-binding protein expression and localization during gastrulation, neurulation, and organogenesis.

Retinoic acid induced heparin-binding protein (RIHB) is a highly basic, soluble polypeptide of the chick embryonic extracellular matrix. We have examined the expression and localization of RIHB during very early embryogenesis by in situ hybridization and immunohistochemistry. RIHB mRNA is very weakly detectable above background in the blastodiscs of unincubated eggs. The expression increases greatly over the first 24 hours of incubation, and is observed throughout the blastodisc in all three of the germ layers following gastrulation. As neurulation occurs, the expression becomes more restricted to certain areas, notably the ectoderm, the neural folds, and especially the notochord. After the neural tube has formed the expression in the tube itself decreases dramatically, whereas the expression in the head ectoderm and the notochord persists. After 72 hours of incubation expression remains relatively high throughout most of the embryo, with higher levels of expression in regions undergoing organogenesis and lower levels in organs which have already differentiated. RIHB protein is also weakly detectable in unincubated eggs as patches of immunoreactive material between the blastodisc and the vitelline. After 6 hours of incubation small regions of basement membrane are immunoreactive. RIHB is detected in this matrix, apparently before even fibronectin. The amount of RIHB protein increases dramatically over the first 24 hours of incubation. It is found in basement membrane separating the epiblast from the hypoblast, then later in that separating the ectoderm from the mesoderm. It is also detected surrounding individual cells, especially of the ectodermal layer. During neurulation RIHB is observed in the basement membrane surrounding the neural fold and the notochord, and in the lamina separating the ectodermal, mesodermal, and endodermal layers. Later in development, RIHB is detected in the basement membrane under the epidermis, throughout the developing limbs, and in the lamina of various developing organs, such as the eye, the pulmonary bud, the intestine, and the mesonephros. These results demonstrate that RIHB is highly expressed during the early embryonic period, by all three germ layers, and is an important and very early component of the embryonic extracellular matrix. Its very broad expression and localization argue for a more general role in development than its demonstrated weak neurotrophic activity.

Immunochemical studies on rabbit calcitonin.

Cross reaction studies using radioimmunoassays specific for human and porcine calcitonin showed that rabbit calcitonin is structurally more closely related to human than to porcine calcitonin.

[Heterogeneity of immunoreactive calcitonin in the plasma of patients with bone marrow cancer]. / Hétérogénéité de la calcitonine immunoréactive dans le plasma de sujets avec cancer médullaire

Plasma from patients with medullary carcinoma containing very high levels of immunoreactive calcitonin were fractionated by filtration on Sephadex gel. In all cases the elution gave four immunoreactive fractions. Two of these fractions correspond to the volume of elution of the monomere and of the dimere of human calcitonin. The two other fractions emerge at an elution volume corresponding to much higher molecular weights. After stimulation of calcitonin secretion in vivo, by dynamic tests, the fractions corresponding to the monomere and dimere increase more strongly than the two other fractions. Preliminary studies of secretion, in vitro, of calcitonin by medullary carcinoma tissue, show the presence in the incubate of four immunoreactive forms having the same elution characteristics as those found in the plasma. The significance of these results is discussed.