Production and properties of a monoclonal antibody against human epidermal growth factor

A monoclonal antibody against human epidermal growth factor (hEGF) was obtained from a mouse hybridoma cell line. The purified monoclonal antibody from the ascites fluid of a mouse injected with one of the cell lines was specific for hEGF and did not cross-react with mouse EGF (mEGF). Its Kd value for hEGF was 1.4 X 10(-9) M. This monoclonal antibody inhibited the biological activities of hEGF, including its binding to the receptor of BALB/3T3 cells and its stimulation of DNA synthesis in the cells, but did not affect the activities of mEGF. The monoclonal antibody completely inhibited DNA synthesis stimulated by human urine from a patient without a tumor, but only partially inhibited the stimulatory activity in urine from a tumor-bearing patient.     

Amino-Terminal Charge Affects the Periplasmic Accumulation of Recombinant Heregulin/EGF Hybrids Exported Using theEscherichia coliAlkaline Phosphatase Signal Sequence

AnEscherichia coliexpression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA–hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated byin vitroreplacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein inE. coliperiplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed inE. coliusing the PhoA signal sequence for protein export.     

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.     

Age-related decrease of urinary excretion of human epidermal growth factor (hEGF)

Epidermal growth factor (EGF) has been first isolated from the submandibular glands of the male mouse and recently from human urine. Despite its potent mitogenic effect in a variety of tissues, the physiological functions of EGF in human still remain undetermined. In order to study the effect of age on urinary human EGF (hEGF), we have evaluated urinary excretion of hEGF in normal subjects over a wide range of age (20–79 yr.) using homologous radioimmunoassay (RIA) for hEGF. Urinary excretion of hEGF expressed as a function of creatinine significantly decreased with increasing age, age, while females excreted significantly more hEGF than males. These data suggest that urinary excretion of hEGF decreases with age in normal subjects which may be due to reduced synthesis and/or secretion of hEGF.     

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β- -thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.     

Synthesis and secretion of an hEGF-like immunoreactive factor by human gastric cancer cells (MKN-45)

A large amount of an immunoreactive factor was detected in the medium conditioned by human gastric cancer cells, strain MKN-45, by our enzyme immunoassay system for human epidermal growth factor (hEGF) based on hEGF isolated from urine. However, the dose-response curve of the immunoreactive factor (designated as MKN-45 EGF) was not parallel with the standard curve of hEGF. The molecular weight of MKN-45 EGF was slightly larger than that of hEGF and was estimated to be 7,000-8,000 by gel filtration on Sephadex G-50. On isoelectric focusing analysis, MKN-45 EGF gave a major peak at pH 5.0 and a minor one at pH 4.3. These results demonstrate that MKN-45 cells synthesize and secrete into the culture medium a polypeptide immunologically related to hEGF.     

HIV-1 Tat peptide immunoconjugates differentially sensitize breast cancer cells to selected antiproliferative agents that induce the cyclin-dependent kinase inhibitor p21WAF-1/CIP-1

In this study, we evaluated the ability of anti-p21 antibodies conjugated to 17-mer peptides [GRKKRRQRRRPPQGYGC] harboring the membrane-translocating and nuclear import sequences [underlined] of HIV-1 tat protein to inhibit the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1) (p21) and differentially sensitize MDA-MB-468 and MCF-7 human breast cancer (BC) cells to the antiproliferative effects of treatments that induce or do not induce p21. BC cells were treated with increasing concentrations of epidermal growth factor (EGF; 0.5-10 nM), the topoisomerase I inhibitor, camptothecin (CPT; 0.1-4 muM), or increasing doses of gamma-radiation (2-20 Gy). Western blot was used to evaluate p21 expression. The effect of treatment on cell cycle distribution was studied. Growth inhibition was measured by the WST-1 assay. Expression of p21 was increased in MDA-MB-468 cells treated with EGF or CPT but not by gamma-irradiation. MCF-7 cells exhibited p21 upregulation following exposure to CPT and gamma-radiation but not EGF. EGF caused cell cycle arrest in G(1) phase for MDA-MB-468 cells. CPT caused G(1)-phase arrest in MDA-MB-468 cells and prolonged S phase in MCF-7 cells. gamma-Radiation caused an increase in cells in G(2)/M phase for MDA-MB-468 and MCF-7. MDA-MB-468 cells were growth-inhibited by EGF, CPT, and gamma-radiation. MCF-7 cells were growth-stimulated by EGF and inhibited by CPT and gamma-radiation. Combining EGF with tat-anti-p21 immunoconjugates (ICs) amplified the growth-inhibitory effect on MDA-MB-468 cells 1.2-fold to 2.3-fold, but had no effect on the growth stimulation of MCF-7 cells by EGF. Tat-anti-p21 ICs sensitized MCF-7 cells 1.4-fold to gamma-radiation but had no effect on the growth of gamma-irradiated MDA-MB-468 cells. Tat-anti-p21 ICs sensitized both MDA-MB-468 and MCF-7 cells 1.7-fold to CPT. We conclude that tat-anti-p21 ICs are promising sensitizers for cytotoxic cancer therapies and that their sensitization is dependent on treatment-related p21 expression. This general approach could potentially be extended to other growth-regulatory molecules that are associated with tumor growth and progression.     

Human breast cancer cells synthesize and secrete an EGF-like immunoreactive factor in culture

A human breast cancer cell line, strain MCF-7, in culture synthesized and secreted a large amount of a polypeptide (designated as MCF-7 EGF) immunologically related to human epidermal growth factor (hEGF). The molecular weight of MCF-7 EGF estimated by gel filtration on Sephadex G-75 was similar to that of hEGF from human urine. On isoelectric focusing analysis, MCF-7 EGF gave a major peak at pH 4.6 and a minor peak at pH 5.0. In our enzyme immunoassay system, however, the dose-response curve of MCF-7 EGF did not show good parallelism with that of standard hEGF. From these results, it is suggested that MCF-7 cells synthesize and secrete a polypeptide immunologically related to hEGF into the culture medium.     

Expression of a fusion protein containing human epidermal growth factor and the collagen-binding domain of <Emphasis Type="Italic">Vibrio mimicus</Emphasis> metalloprotease

Human epidermal growth factor (hEGF) is a polypeptide of 53 amino acids, is an important autocrine/paracrine factor in the human body, and is used in the pharmaceutical and cosmetics industries. We constructed a fusion hEGF protein with a collagen-binding domain (CBD) composed of 33 amino acids from Vibrio mimicus metalloprotease (VMCBD). The CBD segment of the metalloprotease was fused at the C terminus of the hEGF protein. The recombinant fusion protein was expressed in Escherichia coli and purified. The purified hEGF protein promoted greater growth of human/A-431 cells than did the control hEGF. The fusion EGF protein also showed collagen-binding activity with type I collagen. In contrast, hEGF did not bind to type I collagen. These results suggest that recombinant hEGF protein fused to VMCBD may be able to remain for a long period at injured epidermal tissue acting as a healing agent.     

Semiquantitative immunoblots of membrane protein-epidermal growth factor

Membrane proteins or cytokines are sometimes difficult to isolate and purify. Our group recently concentrated on epidermal growth factor (EGF) protein expression studies. Mature EGF was initially identified from mouse submaxillary gland extract as a stimulator of eyelid opening and incisor eruption when injected into newborn mice and rats. The EGF precursor is a transmembrane protein with eight additional EGF-like repeats. Our previous study has shown that the EGF precursor without these eight EGF-like repeats (hEGF) was biologically active. Here, we introduce a modified method for rapid detection of hEGF. The membranous protein was directly extracted from various organs of transgenic mice (including the submandibular gland, kidney, liver, heart, and testis) with two different buffers and easily detected by semiquantitative immunoblotting.     

Identification of a determinant of epidermal growth factor receptor ligand-binding specificity using a truncated, high-affinity form of the ectodomain

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg(45) in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-alpha with high affinity (K(D) = 13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu(367), Gly(441), or Glu(472) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab528. However, mutant Gly(441)Lys showed markedly reduced binding to hEGF, implicating Gly(441), in the L2 domain, as part of the binding site that recognizes Arg(45) of hEGF.     

Cell selective targeting of a simian virus 40 virus-like particle conjugated to epidermal growth factor

Simian virus 40 (SV40) virus-like particles (VLPs) are efficient nanocarriers for gene delivery. VLPs conjugated to human epidermal growth factor (hEGF) were prepared and the cell selectivity of the VLP was examined using human epithelial carcinoma A431 cells, which overexpress the EGF receptor. The endocytic efficiency was determined by the level of Gaussia luciferase activity from the encapsulated protein in hEGF-conjugated VLPs. EGF receptor-mediated endocytosis of hEGF-conjugated VLPs was significantly increased and was confirmed by fluorescence imaging using mCherry encapsulated in hEGF-conjugated VLPs. These results suggest that VLPs of SV40 conjugated to a specific ligand could be used for cell selective gene delivery.     

Epidermal growth factor and insulin-like growth factor-1 preserve cell viability in the absence of protein synthesis

Summary Prolonged exposure of cells to the potent protein synthesis inhibitor cycloheximide (CHX) terminates in cell death. In the present study we investigated the effect of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin on cell death induced by CHX in the human cancerous cell lines MDA-231 and MCF-7 (breast), KB (oral epidermoid), HEP-2 (larynx epidermoid), and SW-480 (colon), and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHX was shown to induce cell death in a concentration (1 to 60 μg/ml) and time (24 to 72 h)-dependent manner in each of the five cell lines. EGF at physiologic concentrations (2 to 40 ng/ml) reduced cell death close to control level (without CHX) in the cell lines HEP-2, KB, MDA-231, and SW-480, but had almost no effect on cell death in the MCF-7 cells. IGF-1 at physiologic concentrations (2 to 40 ng/ml) reduced cell death nearly to control level in the MCF-7 cells, but had only a partial effect in the other four cell lines. Insulin at supraphysiologic concentration (10 000 ng/ml) mimicked the effect of IGF-1 in each of the cell lines. CHX at concentrations that induced about 60% cell death, inhibited about 90% of protein synthesis as measured by [³H]leucine incorporation. Protein synthesis remained inhibited although cell viability was preserved by EGF or IGF-1. These results indicated that the mechanism by which EGF or IGF-1 preserve cell viability does not require new protein synthesis and may be mediated via a posttranslational modification effect.     

Cloning and expression of the ccdA-associated thiol-disulfide oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production

Brevibacillus choshinensis (Bacillus brevis) is a protein-hyperproducing bacterium with a useful host-vector system for the production of recombinant proteins. Here, we cloned the ccdA-catA (cmacr;cdA āssociated thioredoxin-like tmacr;hiol-disulfide oxidoreductase) locus of B. choshinensis HPD31-S5. CatA protein (molecular weight, 19664) contains a thioredoxin-like motif, Cys-Gly-Pro-Cys. It was successfully expressed in B. choshinensis extracellularly ( approximately 100 microg x ml(-1) culture) using the secretion vector pNCMO2, and in Escherichia coli intracellularly ( approximately 350 microg x ml(-1) culture) with an amino-terminal His-tag. Both recombinant proteins showed thiol-disulfide oxidoreductase activity. Incubation of non-native human epidermal growth factor (hEGF) containing incorrect disulfide bonds with B. choshinensis cells secreting CatA protein resulted in the stimulation of the conversion of non-native hEGF to the native form. Furthermore, co-expression of CatA protein with recombinant hEGF in the B. choshinensis production system increased the yield of native hEGF.     

Mitogen limitation and bone morphogenetic protein-4 promote neurogenesis in SFME cells, an EGF-dependent neural stem cell line

Serum-free mouse embryo (SFME) cells are an epidermal growth factor (EGF)-dependent established line derived from brains of 16-d-old Balb/c mouse embryos. SFME cells grow indefinitely in serum-free medium without replicative senescence, chromosomal abnormalities, or malignant transformation. SFME cells express nestin, a neural stem cell marker, under serum-free conditions. Exposure to serum or transforming growth factor β (TGF-β) leads to a marked increase in differentiation toward the astrocytic lineage with expression of glial fibrillary acidic protein and other astrocyte markers. In this study, we show that treatment of SFME cells with bone morphogenetic protein-4 (BMP-4), another member of the TGF-β family, led to differentiation toward a neuronal lineage under conditions of low mitogenic stimulation (0.5 ng/mL) by EGF and fibroblast growth factor. Maximum mitogenic stimulation with 50 ng/mL EGF blocked the BMP-4 effect on neuronal differentiation, but did not block TGF-β-induced expression of markers of the astrocytic lineage. BMP-4 treatment also enhanced the activity of the neuron-specific enolase (NSE) promoter in SFME-NSE-lacZ cells that carry the gene for bacterial β-galactosidase under the control of the NSE promoter. Extended BMP-4 treatment caused SFME cells to express a neuronal phenotype synthesizing gamma-aminobutyric acid. These results indicate that SFME cells have the capacity to generate both neurons and astrocytes in vitro, which resemble the behavior of EGF-dependent multipotential stem cells in the central nervous system, and establish a relationship between effects of BMP-4 and degree of mitogenic stimulation by other peptide growth factors.     

转基因迷你番茄及其对酒精引起的胃伤害的保护作用

表皮生长因子(EGF)具有促进多种细胞增殖的作用, 尤其在维持消化道黏膜完整和促进消化道溃疡愈合方面作用巨大。以前的研究多集中在细菌和酵母中表达, 本研究试图以番茄作为生物反应器生产重组人 EGF, 使之成本降低, 应用方便。依据人 EGF 的基因序列, 设计合成了番茄密码子偏爱的人 EGF 基因, 并将其构建到植物表达载体 pCAMBIA2300 中, 通过农杆菌介导得到了含有人 EGF 基因的转基因番茄。放射免疫法检测到每克鲜重果实中的表达量达 3.48 ± 1.01 ng。将果汁灌喂小白鼠 15 天(相当于每鼠每天喂服 24 ng rhEGF)能显著抵抗酒精引起的胃溃疡形成, 溃疡指数由 42.20 ± 18.13 下降为 16.25 ± 9.57。     

Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.

NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function.     

Epidermal growth factor alters metabolism of inositol lipids and activity of protein kinase C in mouse embryo palate mesenchyme cells

Epidermal growth factor (EGF) stimulated mouse embryo palate mesenchyme (MEPM) cells (1) to incorporate [32P]O4(3-) into phosphatidylinositol (PI), phosphatidylcholine, and phosphatidic acid over a period of 60 min; 2) to incorporate [32P]O4(3-) into polyphosphoinositides as a function of time; and 3) to incorporate [32P]O4(-3) into PI, only, as a function of concentration when the period of stimulation was kept short. EGF stimulated the release of radiolabeled inositol phosphates from MEPM cells that had been radiolabeled with [3H]myoinositol. The release of inositol 1-phosphate was sustained over a period of at least 60 min, whereas the release of inositol 1,4-bisphosphate and inositol trisphosphate peaked during the first 10 min of stimulation. EGF also stimulated phosphorylation of an Mr 80,000 protein whose pI, phosphopeptide map, and phosphoamino acid pattern were identical to those of an Mr 80,000 protein phosphorylated in response to phorbol 12-myristate 13-acetate. Mobilization or metabolism of arachidonic acid was not stimulated under the same conditions that permitted EGF to alter inositol lipid metabolism. We interpret these data to mean that 1) in contrast to the findings with some cell lines, alterations in inositol lipid metabolism may be part of the signalling mechanism for EGF in embryonic cells; 2) EGF is capable of activating inositol-dependent signalling pathways leading to activation of protein kinase C in MEPM cells; and 3) mobilization and metabolism of arachidonic acid are not an inherent part of this signalling mechanism.     

Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor

Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32P]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32P] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32P] incorporation into threonine-654 reached 50% of the [32P] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na+/H+ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H+ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C activity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function.