Polypeptide Factors Research Articles

4963485 Brain derivable polypeptide factors and antibodies thereto: Mohamme Shoyab, Hans Marquardt, George J Todaro assigned to Oncogen

4963485 Brain derivable polypeptide factors and antibodies thereto: Mohamme Shoyab, Hans Marquardt, George J Todaro assigned to Oncogen

A polypeptide factor produced by fibrosarcoma cells that induces endothelial tissue factor and enhances the procoagulant response to tumor necrosis factor/cachectin.

Intravascular clot formation, localized to the neoplasm, is an early component of the vascular response to tumor necrosis factor (TNF)/cachectin. Fibrin is closely associated with the endothelial cell surface, and multiple microthromboses lead to reduced blood flow in the tumor. We have identified a tumor-derived mediator which enhances endothelial procoagulant activity and the cellular response to TNF using cultured cells derived from a murine methylcholanthrene A (meth A)-induced fibrosarcoma as a model system. A heat-stable protease K-sensitive polypeptide, Mr approximately 44,000 on nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 56,000 reduced), was purified approximately 500,000-fold from serum-free culture supernatants of meth A cells by sequential Q-Sepharose, Mono S, reversed phase, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on immunologic criteria, biologic activity, and other molecular properties, meth A factor appears to be distinct from other cytokines and growth factors. Purified meth A factor induced transcription of the tissue factor gene and expression of procoagulant activity by cultured human endothelium (half-maximal effect for the latter at approximately 6-8 pM). Furthermore, co-incubation of endothelium with meth A factor together with TNF enhanced induction of tissue factor in a more than additive manner. These data indicate that certain tumors elaborate an apparently unique molecule which can alter hemostatic properties of the vessel wall, potentially modulating reactivity of the tumor vasculature to host response mediators.

Growth regulation of A431 cells. Modulation of expression of transforming growth factor-alpha mRNA and 2',5'-oligoadenylate synthetase activity.

To explore bidirectional regulatory interactions between interferons and autocrine polypeptide factors, we examined the modulation of expression of transforming growth factor-alpha and 2',5'-oligoadenylate synthetase activity in A431 epidermoid carcinoma cells after treatment with interferon-gamma and transforming growth factor-alpha. Treatment of A431 cells with interferon-gamma increased steady state levels of transforming growth factor-alpha mRNA by 4-fold and increased the levels of transforming growth factor-alpha in the culture medium. There were additive growth inhibitory effects upon coaddition of exogenous transforming growth factor-alpha and interferon-gamma to the cultures. Addition of transforming growth factor-alpha to A431 cell cultures in the absence of interferon could stimulate the induction of 2',5'-oligoadenylate synthetase activity by more than 2-fold. These findings demonstrate that the induction of transforming growth factor-alpha in interferon-gamma-treated A431 cells could act to regulate interferon-induced gene(s), e.g. 2',5'-oligoadenylate synthetase, suggesting interactions between a potential autocrine growth factor and the interferon system in the growth regulation of A431 cells.

Corneal Endothelium Modulation Factor Released by Polymorphonuclear Leukocytes

Polymorphonuclear leukocytes produce a polypeptide factor that is released into the medium. This factor is partially purified 83-fold by ammonium sulfate precipitation followed by chromatography on a DEAE-Sephadex or heparin-Sepharose column. The partially purified factor is trypsin-sensitive. This factor affects a population of rabbit corneal endothelial cells by modulating them to fibroblastlike cells and by further stimulating their growth, leading to the formation of colonies of multilayered modulated cells. There is a dose-dependent phenotypic modulation of corneal endothelial cells by the partially purified cornea! endothelium modulation factor (CEMF); cell shape is changed and type I collagen synthesis is increased with greater concentrations of CEMF. Since the fully modulated endothelial cells have collagen phenotypes distinct from those of normal cells, collagen synthesized by the first-passaged cells (a mixture of normal and modulated cells) was determined by immunoblot analysis with antibodies specific against types I and IV collagens. The first-passaged cells, in the presence of CEMF, contained a large amount of type I collagen (modulated phenotype) and a dramatically reduced amount of type IV collagen (physiologic type), whereas the normal endothelial cells demonstrated strongly positive staining only with antibodies to type IV collagen. Using cloned cDNA probes, the relative quantities of the transcripts of these collagens were determined by slot-blot hybridization; the first-passaged cells contained type IV collagen RNA in an amount similar to the normal cells, but a slightly larger amount of type I mRNA. These results demonstrate a functional involvement of a protein factor released by polymorphonuclear leukocytes in modulating cell shape and collagen gene expression in corneal endothelial cells. Invest Ophthalmol Vis Sci 31:313322,1990

Prostanoids as second messengers of polypeptide growth factors

Prostaglandin H synthase (PGHs), also known as cyclooxygenase, is an unstable enzyme whose mRNA has an half life of 10 minutes. Some polypeptide factors have been reported to induce the enzyme in target cells. We have purified and characterized a component of animal sera which behaves as a potent inducer of human monocyte PGHs. This factor, called serum monocytotropic factor, has been identified in human platelets and it appears to be structurally and biochemically different from identified platelet factors, such as platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), while showing strong similarities to colony stimulating factor 1 (CSF-1), so far undetected in platelets. Moreover, we have shown, by immunoblot analysis, that CSF-1 behaves as a potent and specific inducer of monocyte PGHs. The hypothesis that prostanoids may be considered as second messengers of platelet CSF-1 like factor, as well as of other growth factors and that PGHs induction plays a pivotal role in this process, will be illustrated.

Growth factors and oncogenes in breast cancer

The growth of normal breast epithelial cells is regulated by a complex interacting system of polypeptide factors and by steroid hormones. The cells respond to these factors through receptors which generate mitogenic and other intracellular signals. These second messengers provoke complex responses which may ultimately result in DNA replication and cell division. A comparison of normal cells and tumour cells, either in culture or from primary tumour biopsies, has revealed differences in growth factor and growth factor receptor expression. Such changes may represent aspects of the process of malignant transformation. In addition some evidence suggests that changes in second messenger systems may also occur. Finally several changes in nuclear oncogenes have been observed in breast cancers. It has been proposed that changes in the nuclear oncogenes, perhaps involving the loss of function of tumour suppressor genes, may allow cells to enter the cell cycle. Changes in growth factors, their receptors or intracellular second messenger systems may stimulate unregulated growth. The combination of these events provide a model for the process of carcinogenesis.

Effect of low-molecular-weight factors os thymus and pineal gland on life span and spontaneous tumour development in female mice of different age

Female SHR mice, aged 3.5 or 12 months, were exposed monthly to 5-day long courses of subcutaneous injections of 0.1 mg thymus-derived or pineal gland-derived polypeptide factors (TF and PF, respectively) or 0.9% sodium chloride solution (control). PF treatment increased life span of both young and midelle-aged mice by 20% and 17%, respectively, and TF increased the life span only in young mice. Both factors when administered to young mice caused a decrease in both overall tumour incidence and incidence of mammary adenocarcinomas (TF, 1.8-fold decrease; PF, 2.6-fold decrease). TF administration to mature mice did not produce any antitumour effect, whereas PF possessed certain anti-tumour activity, but the response was far less pronounced than in young animals. The results obtained give additional evidence of the geroprotective and anti-tumour effect of thymus and pineal gland-derived peptide factors. The mechanisms of action of TF and PF and perspectives of clinical use of these agents as geroprotectors are discussed.

Identification of the two types of specific receptor for activin/EDF expressed on friend leukemia and embryonal carcinoma cells

Activin and inhibin are polypeptide factors which control the release of follicle stimulating hormone(FSH) from pituitary cells. The recent finding that erythroid differentiation factor(EDF) is identical to activin showed the multifunctional feature of this protein. To identify the specific receptor for activin, the binding of 125I-labeled activinA was investigated for a number of culture cell lines. Friend leukemia cell, which can be differentiated by activin, and embryonal carcinoma(EC) cells(PCC3, P19 and F9), were found to express 3500–20,000 per cell of activin receptors. Scatchard plot analysis of the binding data shows that the receptors on those cells could be divided into two groups with different Kd values. The Kd values of high and low affinity receptors are 0.15–0.4nM and 1.5–3.0nM respectively. The proportion of the number of the high and low affinity receptors was varied in each cell. Inhibin was able to compete for activin binding to both types of receptors, although the binding affinity was about 50–200 fold lower than that of activinA. Transforming growth factor-β had no binding ability to the activin receptors.

Mast cell lines produce lymphokines in response to cross-linkage of FcεRI or to calcium ionophores

The cross-linkage of high affinity Fc epsilon receptors (Fc epsilon RI) on mast cells and basophils is central to the induction of allergic inflammatory responses. As a result of such cross-linkage, mast cells secrete a variety of preformed biologically active substances, such as histamine, and newly synthesized arachidonic acid metabolites. Here we show that cross-linkage of Fc epsilon RI on a series of nontransformed murine mast cell lines, or treatment of these cells with calcium ionophores, stimulates increased messenger RNA levels and secretion of a group of lymphokines classically produced by a subset of murine T cell lines (TH2 cells). These factors include interleukin-3 (a mast cell growth factor)s interleukin-4 (an IgE 'switch factor'), interleukin-5 (an eosinophil differentiation factor) and interleukin-6 (a factor controlling immunoglobulin secretion). The production of these polypeptide factors by mast cells may have great importance in the induction of allergic and anti-parasite inflammatory responses.

Induction of interleukin‐1 receptors on chondrocytes by fibroblast growth factor: A possible mechanism for modulation of interleukin‐1 activity

Interleukin-1 is a polypeptide factor with profound effects on several cell types, such as chondrocytes, fibroblasts, and T-cells. The ability of interleukin-1 to induce the synthesis of matrix-degradative enzymes, as well as prostaglandin E2, suggests a pivotal role for this mediator in chronic inflammation. Previous studies have shown that the effect of human monocyte interleukin-1 on the synthesis of collagenase and neutral proteases by chondrocytes was enhanced by basic fibroblast growth factor. Using recombinant human interleukin-1B, we have examined whether the potentiation of interleukin-1 effects by fibroblast growth factor is related to changes in the number or affinity of interleukin-1 receptors. Our studies confirm that rabbit articular chondrocytes in culture contain a single class of high-affinity receptors for interleukin-1 with a Ka of 0.9-1.1 x 10(-13) M-1. While the untreated chondrocytes contain approximately 1,620 receptors per cell, fibroblast growth factor-treated cells exhibit a higher number of receptors (approximately 2,960 per cell) with no apparent change in the affinity. The increase in receptor number can be abolished by inhibitors of lysosomal function, indicating a requirement for intracellular processing of the fibroblast growth factor. Our results suggest that the potentiation of interleukin-1 catabolic effects by fibroblast growth factor may be related to its ability to induce additional interleukin-1 receptors on the chondrocyte cell surface.

Den Induced Liver Carcinogenesis in Rats: Growth Inhibitory Polypeptides Isolated from Normal Livers and Liver Tumours

AbstractLow molecular weight partially purified inhibitory polypeptides were isolated from normal livers and liver tumours induced with DEN. Both the polypeptide fractions caused inhibition of 3H-Thymidine incorporation into primary hamster dermal fibroblasts. The polypeptide fractions were effective at as low a concentration as 10 μg/ml tested and the percentage of inhibition increased with increase in the concentration, attaining about 75-80% inhibition at 40 μg/ml concentration. SDS-PAGE and molecular sieve HPLC-analysis showed presence of four polypeptides in both the fractions. It appears that the imbalance between the loss and synthesis of some polypeptide factors with biological activity may be conferring growth advantage during carcinogenesis.

Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides.

Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.

Effects of transforming growth factor type beta upon bone cell populations grown either in monolayer or semisolid medium

Bone has been shown to store large amounts of transforming growth factor type beta (TGF beta) and this has recently been found to be synthesized by bone-forming cells. We report on studies undertaken to examine the effects of platelet-derived TGF beta on different bone cell populations, isolated from 1-day postnatal rat calvaria by sequential enzymatic digestion. In addition, we tried to determine which of these cell populations synthesize TGF beta. In this regard, evidence was collected to indicate that cell populations which were shown to be enriched with osteoblast-like cells synthesize TGF beta. Although the production of the factor appeared to be limited to a particular cell type, its action was found to be of a more general character, as all cell populations were found to respond to TGF beta. Contrary to earlier reports, TGF beta was shown to be inhibitory upon cell proliferation. In this context, growth of cells released during early digestions was reduced considerably more than growth of those released during late digestions. Studies on the effect upon protein synthesis revealed that TGF beta specifically inhibited collagen but not the synthesis of noncollagenous proteins. The synthesis of collagen was altered to a greater extent in cells isolated during late digestions than in cells of the early populations. Further information on the TGF beta-mediated effects on bone cell biology was provided by data showing that both alkaline phosphatase and cAMP production in response to PTH was greatly reduced by TGF beta. Finally, experiments performed to determine whether TGF beta induces any of the bone cell populations to acquire the transformed phenotype revealed that only populations previously shown to be enriched with osteoblast-like cells formed colonies in soft agarose.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of cholinergic retinal neurons from embryonic chicken in monolayer cultures: stimulation by glial cell-derived factors

In recent years evidence has indicated that, like the PNS, the development of the CNS is influenced by neuronotrophic polypeptide factors. In the present study, cultures of dissociated retinal neurons from 8-d-old chicken embryos were used to investigate the role of neuronotrophic factors (NTF) in the development of the neural retina. CAT, which in vivo is located in amacrine cells of the retina, served as a marker for studying the in vitro development of cholinergic retinal neurons. Differentiation of cholinergic cells under control conditions was indicated by a 10-fold increase of enzyme activity during a 7-d culture period. Addition of media conditioned by high-density retinal cultures resulted in a further stimulation of CAT activity by 100-400%. The CAT-stimulating activity was associated with a high-molecular-weight component of the retina conditioned medium (RCM) and was sensitive to protease treatment, but was not affected by other hydrolytic enzymes. The putative cholinergic factor was secreted by retinal cultures virtually free of neurons, suggesting that it is mainly produced by Müller cells. CAT-stimulating activity was also present in extracts from embryonic chicken retinae and medium conditioned by rat retinal cultures. NGF, anti-NGF antiserum, extracts from chicken brain tissues, and a number of other extracts and conditioned media, all known to contain neuronotrophic activities, were found to have no influence on cholinergic development in chicken retinal cultures. An extract from non-retinal eye tissue containing ciliary neuronotrophic factor (CNTF) stimulated CAT activity to the same extent as did RCM.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphokines in the regulation of immune responses

Lymphokines are polypeptide factors produced by lymphocytes and other leukocytes, which modulate leukocyte activities in immune responses. Some of the more important lymphokines are reviewed in detail, with emphasis on those that have been cloned. These include interferon −α and − β, interferon γ, interleukin 2, interleukin 1, tumor necrosis factor and lymphotoxin, B lymphocyte growth factors, hemopoietic growth factors, and others.

Secretion and properties of a polypeptide factor generated by phorbol ester stimulation of human blood platelets

Secretion and properties of a polypeptide factor generated by phorbol ester stimulation of human blood platelets

Effect of polypeptides from the thymus, bone marrow, and bursa of fabricius on immunogenesis and hemostasis in neonatally thymecotimized and antenatally bursectomized chickens

The effect of polypeptides from the thymus, bone marrow and bursa of Fabricius on immunogenesis and hemostasis was investigated in neonatally thymectomized and antenatally bursectomized chickens. It has been established that polypeptide factors from the bursa of Fabricius have the most pronounced effect on the immunity and hemostasis.

INCREASED EPIDERMAL GROWTH FACTOR CONCENTRATIONS IN DONAHUE'S SYNDROME (LEPRECHAUNISM)

Epidermal growth factor (EGF) is a well characterized polypeptide factor known to be important in growth and proliferation of multiple cell types. We have described a patient (Lep Ark I) with Donahue's syndrome whose phenotypic characteristics, i.e., severe acanthosis nigricans, hirsutism, pachyderma, precocious development of teeth and breast buds, and gut hypertrophy are consistent with hypersecretion or hyperfunction of epidermal growth factor. We previously demonstrated that this patient has increased secretion of epidermal growth factor in her urine (151.9±59.4 ug EGF/gm cr compared to 26.7±4.3 ug/gm in control children) but were unable to quantify plasma concentrations at that time. We have now developed a heterologus, double antibody radioimmunoassay (RIA) based on mEGF to estimate plasma levels of EGF in human plasma. Dilutions of the patient's plasma displace 125I-EGF in a linear fashion producing a line parallel to that of the EGF standards. Mean plasma concentration levels of EGF in normal adults by this method are 4.8±1.7 (N=5) and in children 8.4±0.3 (N=5). In contrast, the mean plasma concentration of EGF in Lep Ark I is 20.2±3.3 ng/ml (N=4). Reported blood levels of EGF in humans have ranged from 273±46 pg/ml to 1.89±0.5 ng/ml. Our mean plasma concentrations for children were higher than those previously reported but were less than half the value found in Lep Ark-I. We conclude that the elevated urinary excretion of EGF in Lep Ark-I reflects hyper-secretion in this patient, thus providing a disease state for this important growth factor.

Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate.

The proliferation of isolated normal prostate epithelial cells from rat and man is androgen-independent and requires cholera toxin, insulin, dexamethasone, epidermal growth factor (EGF) and one or more polypeptide factors that are concentrated in bovine neural tissue. The active agents in the neural tissue extract are heparin-binding polypeptides (prostatropins), the predominant form of which has a molecular weight of 17400 and an acetylalanine at the aminoterminus. Prostatropins supported a half-maximal increase in normal prostate epithelial cell number at 50 picomolar. The proliferation of primary and serially-cultured epithelial cells from androgen-responsive Dunning R3327 rat prostate tumors was also androgen-independent, but exhibited dramatic alterations in response to hormones that stimulated normal cell proliferation. At low cell density, androgen-independent growth of isolated tumor-derived epithelial cells was independent on cholera toxin, was stimulated by dexamethasone, required insulin and either EGF or prostatropin. The presence of either EGF or prostatropin masked the response to the other factor. In the absence of EGF, purified prostatropins supported a half-maximal increase in tumor cell number at 7 picomolar. Endogenous production of EGF-like and prostatropin-like factors or both was suggested by the reduced requirement for EGF and prostatropin at high prostate tumor cell density. These results suggest that anti-hormonal therapies against prostate tumor growth should be based on intervention with the activity of insulin (or insulin-like factors) or simultaneous intervention with both EGF and prostatropin (or their homologues).

Use of vaccinia virus to express biopharmaceutical products.

Recent technological advancements have fostered the continued development of vaccinia virus as an efficient eukaryotic cloning and expression vector system. Genetically engineered vaccinia virus strains have been constructed for use (i) as recombinant vaccines for the prophylaxis of infectious disease, (ii) in producing significant quantities of biologically active polypeptide factors or enzymes, and (iii) as basic research tools with which to investigate primary structure-function relationships between proteins and their catalytic activities. This review examines the basic vaccinia vector system, its advantages and limitations, and current areas of research. As a specific example of the power and utility of this approach, attention is focused on the application of this technology to the field of neurobiology, specifically the use of recombinant vaccinia to study the expression, processing, and transport of cellular neuropeptides.