cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumour cell lines

The amino-acid sequence of the precursor of the human tumour cell line-derived platelet-derived growth factor (PDGF) A-chain has been deduced from complementary DNA clones and the gene localized to chromosome 7. The protein shows extensive homology to the PDGF B-chain precursor. Expression of the PDGF A-chain gene is independent of that of the PDGF B-chain in a number of human tumour cell lines, and secretion of a PDGF-like growth factor of relative molecular mass 31,000 correlates with expression of A- but not B-chain messenger RNA.     

Possible positive autocrine feedback in the prereplicative phase of human fibroblasts

The growth of normal diploid fibroblasts is generally thought to be tightly controlled by exogenous growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). Subversion of a growth factor pathway at a regulatory point is considered to be a key event in neoplastic transformation and tumorigenesis. Thus, simian sarcoma virus has acquired the gene encoding the B-chain of PDGF and there is direct experimental proof that SSV-transformation is mediated by a PDGF-like growth factor. There is accumulating evidence that PDGF-like molecules are also synthesized and released by certain normal cells, suggesting an important role of cellularly produced PDGF in development and tissue regeneration. We now present evidence that a transient expression of the gene encoding the PDGF A-chain, and the synthesis and release of functional A-chain homodimers, is an early event in the prereplicative phase of normal human foreskin fibroblasts exposed to PDGF or EGF. Since these cells are PDGF-responsive, the results imply the existence of a positive autocrine signal that may serve as an amplifier of the mitogenic response under certain conditions.     

A role for VEGF as a negative regulator of pericyte function and vessel maturation

Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively. PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators. Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rbeta signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rbeta and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rbeta/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.     

Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells.

Normal cells in culture have membrane receptors for epidermal growth factor (EGF); EGF stimulates cells to divide by binding to these receptors. Cells transformed by murine and feline sarcoma viruses rapidly lose the ability to bind EGF, whereas cells transformed by the DNA tumour viruses, polyoma and SV40, or infected with non-transforming RNA tumour viruses have normal levels of functional EGF receptors. The results suggest that a product of the sarcoma virus genome specifically changes cell EGF receptors; the sarcoma gene product may, then, be functionally related to EGF.     

Transformation of NIH 3T3 cells by a human c-sis cDNA clone

The mechanism of leukaemogenic transformation by human T-cell leukaemia/lymphoma virus (HTLV), a retrovirus implicated in the aetiology of certain adult T-cell leukaemias and lymphomas, is unknown but is conceivably associated with the expression of the cellular analogues of retroviral oncogenes. The HUT-102 cell line, derived from a cutaneous T-cell lymphoma and infected with HTLV, expresses several cellular oncogenes. It is unusual among haemopoietic cell lines in that one of these is c-sis, the gene from which the oncogene v-sis of the simian sarcoma virus was derived, and perhaps the gene for platelet-derived growth factor (PDGF). To explore the possible role of c-sis expression in HTLV-induced disease, we have obtained cDNA clones of c-sis from HUT-102 cells. Here we describe two such clones and report that one of them transforms NIH-3T3 cells. This is the first example of transformation of NIH-3T3 cells by a human onc gene other than c-ras or Blym, as well as the first demonstration of transformation by a human cDNA clone.     

Human transforming growth factors induce tyrosine phosphorylation of EGF receptors

Cultured cell lines of human tumour origin as well as cells transformed by various RNA tumour viruses secrete low molecular weight polypeptide transforming growth factors (TGFs). In addition to competing with epidermal growth factor (EGF) for binding to its cellular receptor, TGFs can transform morphologically fibroblast and epithelial cells in culture. In view of accumulating evidence that tyrosine phosphorylation activity is associated with the transforming genes of various tumour viruses, we determined whether phosphotyrosine levels were elevated in these human tumour cells. We show here that TGFs produced by human tumour cells induce phosphorylation of specific tyrosine acceptor sites in the 160,000-molecular weight (160 K) EGF receptor.     

Structural alteration of viral homologue of receptor proto-oncogene fms at carboxyl terminus

A role for proto-oncogenes in the regulation and modulation of cell proliferation has been suggested by the findings that the B-chain of platelet-derived growth factor (PDGF) is encoded by the proto-oncogene sis and that the erb-B oncogene product is a truncated form of the epidermal growth factor (EGF) receptor. Furthermore, the product of the proto-oncogene fms (c-fms) may be related or identical to the receptor for macrophage colony-stimulating factor (CSF-1). v-fms is the transforming gene of the McDonough strain of feline sarcoma virus (SM-FeSV) and belongs to the family of src-related oncogenes which have tyrosine-specific kinase activity. Furthermore, nucleotide sequence analysis of the v-fms gene product revealed topological properties of a cell-surface receptor protein. To elucidate the features involved in the conversion of a normal cell-surface receptor gene into an oncogenic one, we have now determined the complete nucleotide sequence of a human c-fms complementary DNA. The 972-amino-acid c-fms protein has an extracellular domain, a membrane-spanning region, and a cytoplasmic tyrosine protein kinase domain. Comparison of the feline v-fms and human c-fms sequences reveals that the proteins share extensive homology but have different carboxyl termini.     

Recombinant human TNF induces production of granulocyte-monocyte colony-stimulating factor

Tumor necrosis factor (TNF) is synthesized by macrophages exposed to endotoxin. It produces haemorrhagic necrosis of a variety of tumours in mice and is cytostatic or cytocidal against various transformed cell lines in vitro, but viability of normal human or rodent cells is unaffected. The role of TNF is unlikely to be restricted to the rejection of tumours. Colony-stimulating factors (CSFs) are required for survival, proliferation and differentiation of haematopoietic progenitor cells. The haematopoietic growth factor known as granulocyte-monocyte colony-stimulating factor (GM-CSF) has the ability to stimulate proliferation and differentiation of normal granulocyte-monocyte and eosinophil stem cells and enhance the proliferation of pluripotent, megakaryocyte and erythroid stem cells. In addition, GM-CSF stimulates a variety of functional activities in mature granulocytes and macrophages, for example inhibition of migration, phagocytosis of microbes, oxidative metabolism, and antibody-dependent cytotoxic killing of tumour cells. We show here that TNF markedly stimulates production of GM-CSF messenger RNA and protein in normal human lung fibroblasts and vascular endothelial cells, and in cells of several malignant tissues.     

Selective inhibition of the anchorage-independent growth of myc-transfected fibroblasts by retinoic acid

Fischer rat 3T3 (FR3T3) fibroblasts transfected with a cellular myc gene can be induced to grow and form colonies in soft agar by treatment either with epidermal growth factor (EGF) alone or with the combination of platelet-derived growth factor (PDGF) and type-beta transforming growth factor (TGF-beta). We now show that induction of anchorage-independent growth by each of these sets of growth factors involves different cellular pathways which can be distinguished by their sensitivity to retinoic acid. Colony formation induced by the combined action of PDGF and TGF-beta is 100-fold more sensitive to inhibition by retinoic acid than is colony formation induced by treatment of the myc-transfected cells with EGF. Moreover, retinoic acid (10(-8) M) is inhibitory for colony growth whenever TGF-beta is present, regardless of whether the effects of TGF-beta are stimulatory, as occurs in the presence of PDGF, or inhibitory, as found in the presence of EGF.     

Developmentally regulated production of platelet-derived growth factor-like molecules

Platelet-derived growth factor (PDGF) is thought to mediate the proliferation of smooth muscle cells in injured arteries, and may be involved in the pathogenesis of atherosclerosis. PDGF-like molecules from non-platelet sources may also play a role in the regulation of cell activity in other circumstances. Transformation of cells by a wide range of oncongenic agents appears to activate a cellular gene encoding a PDGF-like molecule, possibly accounting for the ability of transformed cells to grow without addition of exogenous mitogens. We show here that a molecule (PDGF-c) which can compete with 125I-PDGF for binding to PDGF receptors is secreted by cultured rat aortic smooth muscle cells (rASMC) isolated from 13 to 18-day-old rats (pups) but not from three-month-old animals (adults). Thus, production of PDGF-c appears to be developmentally regulated and may be a factor in the more rapid proliferation of rASMC and synthesis of connective tissue components which occurs during growth of the aorta in vivo.     

Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.     

Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors

The primary structure of the receptor for platelet-derived growth factor (PDGF), determined by means of cloning a cDNA that encodes the murine pre-PDGF receptor, is closely related to that of the v-kit oncogene product and the receptor for macrophage colony stimulating factor (CSF-1). Common structural features include the presence of long sequences that interrupt the tyrosine-specific protein kinase domains of each molecule. The PDGF and CSF-1 receptors also share a characteristic distribution of extracellular cysteine residues. Ubiquitin is covalently bound to the purified PDGF receptor, the human gene for which is on chromosome 5.     

Viral MIPα homologous with human MIP-1α acts on HIV co-receptor CCR5

The function and usage of vMIPa encoded by K6 gene of herpesvirus 8 (HHV8) which has homology with human macrophage protein (MIP) have not been clearly known. In the present note the K6 gene of HHV8 was cloned and transfected into NIH3T3 cells and E. coli cells. Conditional media from the 3T3-transfected cells and K6 product vMIPa from E. coli . Cells were used to perform the experiments of ligand-receptor binding and cellular adhesion with peripheral blood macrophages. The conditional media and the purified vMIPa from E. coli could compete to bind to CCR5 located on macrophages from peripheral blood with I¹²⁵-hMIP-1a chemokine of human. Cellular adhesion showed that the conditional media from transfected cells and the purified vMIPa did not induce the adhesion of macrophages from peripheral blood to ICAM-1. In conclusion, vMIPa encoded by K6 gene of HHV8 can bind to CCR5 of peripheral blood macrophage cells and does not induce their adhesion. This suggests that vMIPa enclosed CCR5, also known as HIV co-receptor, may be used to prevent and treat HIV infection.     

ERK信号转导通路在PDGF诱导的人动脉平滑肌细胞增殖中的作用

了解ERK信号转导通路在PDGF诱导的人动脉平滑肌细胞增殖中的作用.原代培养人脐动脉平滑肌细胞（hUASMC）,取生长正常的细胞分4组：对照组,PDGF（platelet derived growth factor）组,ERK阻断剂组和PDGF＋ERK阻断剂组.继续培养24h后,用免疫细胞化学技术测细胞核内核增殖抗原（PCNA）的表达;用MTT法测细胞的增殖活性.结果显示：1）与对照组相比,PDGF组细胞内PCNA 的表达明显增强,MTT法测得A值也升高（P〈0.01）.2）ERK阻断剂可完全抑制PDGF诱导的PCNA 的表达增多和A值的升高.结论：ERK信号转导通路参与了PDGF诱导的人动脉平滑肌细胞的增殖.     

血小板衍生生长因子PDGF与肿瘤血管生成的研究进展

血小板衍生生长因子（platelet-derived growth factor,PDGF）是由多种细胞产生的重要多肽生长因子,可促进结缔组织细胞（如血管内皮细胞、平滑肌细胞）增殖等。PDGF家族目前已知的有4个成员,即PDGF-A,B,C,D。具有生物活性的PDGF分子一般以二硫键连接的同源二聚体或异源二聚体形式组成。PDGF经其受体PDGFR（platelet-derived growth factor receptor,PDGFR）介导后产生相应的生物学效应。体内外研究证实PDGF与肿瘤血管生成密切相关,PDGF主要通过募集周细胞刺激肿瘤血管生成,且周细胞的募集有利于肿瘤血管的成熟、稳定和存活。靶向PDGF的抗血管生成药物已成为肿瘤血管生成抑制剂中新的重要成员。     

Scatter factor is a fibroblast-derived modulator of epithelial cell mobility

Various factors are known to regulate cell growth and differentiation, but less is known of agents which affect movement and positioning, particularly in epithelial-mesenchymal interactions. Cultured human embryo fibroblasts release a protein with a relative molecular mass (Mr) of approximately 50,000 (50K) that affects epithelial cells by causing a disruption of junctions, an increase in local motility and a scattering of contiguous sheets of cells. To investigate specificity, a range of cells has been examined for the ability to produce the factor and for sensitivity to its action. Most freshly isolated normal epithelia and epithelia from cell lines of normal tissue, but not epithelia from tumour cell lines or fibroblasts, were sensitive to scatter factor. In contrast, production of the factor, as identified by activity and by chromatography, was restricted to embryonic fibroblasts and certain variants of 3T3 and BHK21 cells and their transformed derivatives. We conclude that the scatter factor is a paracrine effector of epithelial-mesenchymal interaction, which affects the intercellular connections and mobility of normal epithelial cells. The factor might be involved in epithelial migration, such as occurs in embryogenesis or wound healing.     

Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.