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.     

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.     

Assessment by a two-site enzyme immunoassay of human epidermal growth factor (urogastrone) in the urine of patients with various gastrointestinal diseases including malignant tumors

By using our two-site enzyme immunoassay (EIA) system, the levels of human epidermal growth factor (hEGF) in the urine of patients with various gastrointestinal diseases including malignant tumors were measured. Urinary excretion of hEGF in patients having undergone gastric resection, expressed as a function of creatinine, was found to be somewhat decreased. While the levels of hEGF in patients with gastric cancer were significantly increased. Then, the molecular features of hEGF in the urine of patients with gastric cancer were examined by gel filtration. The elution profile demonstrated that high molecular weight components, which immunologically cross-reacted with hEGF, were considerably increased. On the other hand, the level of normal EGF with a molecular weight of 6500 was decreased to some extent. These results suggest that processing of the EGF precursor into an active EGF molecule is partially suppressed in patients with gastric cancer.     

Partial purification and characterization of epidermal growth factor in human breast milk

Human epidermal growth factor (hEGF), a potent growth stimulator of many tissues in culture, has been isolated from human urine and subsequently identified in many human biological fluids including breast milk. In this study, partial purification and characterization of hEGF-like substance(s) in human milk were performed using homologous hEGF radioimmunoassay (RIA) and radioreceptor assay (RRA). hEGF-like material(s) was extracted from pooled human milk by ethanol precipitation, followed by adsorption to cation- and anion-exchange resin. DEAE-Sephadex G-25 ion-exchange chromatography of human milk extracts revealed three major components with hEGF activity (peak I, II, III) eluted with a linear gradient by ammonium acetate. The competitive binding curves for these components were parallel to those for standard hEGF in both RIA and RRA. The apparent molecular weight of peak I was approximately 6,500 and that of peak II and III was approximately 7,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The pI value for peak I was approximately 4.5 and that for peaks II and III was approximately 5.0 by isoelectric focusing. These data are comparable to the size and charge heterogeneity of hEGF in human urine extracts. In conclusion, the major components of hEGF in human milk appear to be physicochemically, immunologically and biologically (receptor binding activity) indistinguishable from hEGF of urinary origin.     

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.     

Human term placenta contains transforming growth factors

Growth factors of apparent molecular weights of 6,000, 10,000, 20,000 and one in excess of 30,000 daltons can be isolated from acid-ethanol extracts of human term placentas. Each size class of growth factor resembles transforming growth factor (TGF) in that it stimulates anchorage independent growth of normal rat kidney cells and competes with EGF for binding to EGF membrane receptors. The 6,000, 10,000 and 20,000 molecular weight polypeptides also resemble TGF in their acid and heat stability, and their requirement for intact disulfide bonds for growth promoting activity. By homologous radioimmunoassay, neither the 6,000 nor 10,000 dalton polypeptide is related to human epidermal growth factor (hEGF). The presence of these TGFs in ample concentrations (approximately 100 ng of EGF equivalents per term placenta for the 10,000 dalton polypeptide) indicates the usefulness of this tissue source for study of human TGFs.     

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions

Human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGFalpha) are prototypical of structurally related polypeptide mitogens which interact with the epidermal growth factor receptor (EGFR). Several determinants of receptor recognition that specify function have been proposed on the basis of structural criteria. This study evaluates the role of one such candidate, H16 of hEGF, by site-specific mutagenesis. When assayed for receptor tyrosine kinase stimulation using (Glu4,Tyr1)n as the exogenous substrate in vitro, the relative agonist activities of position 16 mutants range from 14-263% of wild-type hEGF. The rank order of potency was found to correlate with the relative receptor binding affinities of the mutants, which range from 7-272% of wild-type, as determined by radioreceptor competition assays. The mitogenic activity of the H16 mutants is similar to that of wild-type hEGF as determined by clonogenic assays using rat tracheal epithelial cells. While the colony forming efficiencies do not reflect significant differences in growth rate or survival characteristics in the presence of the hEGF variants, it is reduced to 1.6% in control cultures which lack EGF in the medium. The results show that H16 of hEGF, although not essential for mitogenic activity, optimizes receptor recognition by hydrogen-bond donor/acceptor interactions and may share this feature with H18 of hTGFalpha.     

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.     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

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.     

Prolactin can stimulate general protein synthesis in human breast cancer cells (MCF-7) in long-term culture

Prolactin significantly stimulated protein synthesis rates in a human breast cancer cell line, MCF-7. Total protein per culture also increased to 148% of controls. Proteins precipitated from cell homogenates at pH 4.65 in the presence of calcium ions were resolved by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The specific radioactivity for almost every polypeptide band increased as a result of protlactin treatment. Cell-type specificity for this prolactin action was indicated by the failure of two other human cell lines (HBL-100 and HEL) to respond similarly. This report is the first demonstration that prolactin has the capability to stimulate general protein synthesis in human breast cancer cells in long-term culture.     

Some properties of human epidermal growth factor (hEGF)-like immunoreactive material originating from platelets during blood coagulation

During blood coagulation, a considerable amount of human epidermal growth factor (hEGF)-like immunoreactive material (designated as platelet hEGF-LI) was liberated from platelets. The molecular nature of the platelet hEGF-LI was examined. The molecular weight of platelet hEGF-LI estimated by gel filtration was approximately 60,000-70,000. On chromatofocusing chromatography, platelet hEGF-LI was eluted mainly at pH 4.75 as a sharp peak with a minor peak at 4.30, like urine EGF. By treatment with 2-mercaptoethanol, the factor was converted into a material with molecular weight of 35,000-40,000. These results suggest that the majority of hEGF-LI in platelets may exist either in a covalently bound-form with some protein(s) in platelets or as a dimer intermolecularly cross-linked by an S-S linkage. Details of the biological properties and the physiological significance of the platelet hEGF-LI remain to be clarified.     

Overproduction of human epidermal growth factor/urogastrone in Escherichia coli and demonstration of its full biological activities

A man-made gene coding for [21-Leu] human epidermal growth factor (hEGF)/β-urogastrone was constructed, inserted into a lacZ fusion-gene expression vector, and cloned into Escherichia coli. In the cloned cells the β-galactosidase/hEGF fusion gene was efficiently expressed and the yield of the hybrid protein reached 10% of the total cellular protein. The [21-Leu] hEGF synthesized in the bacterial cells was proved to be as functional as the natural hEGF or urogastrone and mouse EGF by the following criteria: (1) stimulation of cell proliferation, (2) stimulation of thymidine incorporation into cultured cells, (3) competition with mouse EGF for binding sites on the plasma membrane of human KB cells, and (4) stimulation of phosphorylation of a membrane-bound protein of human KB cell, which has an apparent molecular weight of 150 000 to 170 000 and is indistinguishable from the protein phosphorylated in the presence of mouse EGF in the sodium dodecyl sulfate—polyacrylamide electrophoretic gel. The hEGF produced in the bacterial cells also resulted in precocious eyelid-opening by the daily subcutaneous injection into newborn mice and in accelerated incisor eruption in the animals as mouse EGF did, indicating that the hEGF is also fully active in vivo or physiologically.     

Receptor-Purified,Bolton-Hunter Radioiodinated,Recombinant, Human Epidermal Growth Factor: An Improved Radioligand for Receptor Studies

Abstract

We report an assessment of the applicability of the Bolton-Hunter method to the radioiodination of epidermal growth factor (EGF). Recombinant human EGF (hEGF) could be radioiodinated successfully by this method, whereas murine EGF could not. Bolton-Hunter ¹²⁵I-labeled hEGF was compared with commercial ¹²⁵I-labeled hEGF prepared by the chloramine-T radioiodination method. Neither radioligand was sufficiently pure for a detailed characterization of the purportedly heterogeneous pattern of binding of EGF to its receptors. A procedure based on receptor adsorption was thus developed for repurification of the Bolton-Hunter ¹²⁵I-labeled hEGF. This provided a much purer radioligand suitable for detailed studies of receptor-binding heterogeneity.     

Purification and characterization of epidermal growth factor (beta-urogastrone) and epidermal growth factor fragments from large volumes of human urine

We purified human epidermal growth factor (hEGF) and hEGF fragments from a benzoic acid precipitate of the materials not adsorbed to silica gel in 330 liters of human urine by a relatively brief, simple, and efficient method employing sequential batch absorption to and stepwise elution from CM-cellulose and DEAE-cellulose, Bio-Gel P-10 chromatography in 50 mM HCl, and three reverse-phase HPLC steps for final resolution and purification of hEGF components. Recovery of hEGF was 29%. Eight apparently homogeneous hEGF components were recovered, each of which had similar activities in a homologous hEGF radioimmunoassay and an EGF radioreceptor assay using human placental membranes. Amino acid composition analysis indicated that there were four pairs of components that represented intact 53-amino acid hEGF, hEGF-(1-52), hEGF-(1-51), and hEGF-(1-50); intact hEGF accounted for one-third of the total materials recovered. Automated Edman degradation of each component for at least 10 cycles revealed a single amino acid sequence identical to that proposed for human beta-urogastrone. Similar immunoreactive hEGF components were observed in similar proportions in freshly voided urine, indicating that they were not artifacts of the purification process. Thus, multiple forms of fully biologically active hEGF (i.e., beta-urogastrone) can be relatively easily and efficiently purified from large volumes of human urine.     

Evolution of distinct EGF domains with specific functions

EGF domains are extracellular protein modules cross-linked by three intradomain disulfides. Past studies suggest the existence of two types of EGF domain with three-disulfides, human EGF-like (hEGF) domains and complement C1r-like (cEGF) domains, but to date no functional information has been related to the two different types, and they are not differentiated in sequence or structure databases. We have developed new sequence patterns based on the different C-termini to search specifically for the two types of EGF domains in sequence databases. The exhibited sensitivity and specificity of the new pattern-based method represents a significant advancement over the currently available sequence detection techniques. We re-annotated EGF sequences in the latest release of Swiss-Prot looking for functional relationships that might correlate with EGF type. We show that important post-translational modifications of three-disulfide EGFs, including unusual forms of glycosylation and post-translational proteolytic processing, are dependent on EGF subtype. For example, EGF domains that are shed from the cell surface and mediate intercellular signaling are all hEGFs, as are all human EGF receptor family ligands. Additional experimental data suggest that functional specialization has accompanied subtype divergence. Based on our structural analysis of EGF domains with three-disulfide bonds and comparison to laminin and integrin-like EGF domains with an additional inter-domain disulfide, we propose that these hEGF and cEGF domains may have arisen from a four-disulfide ancestor by selective loss of different cysteine residues.     

Regulation of epidermal growth factor-receptor by estrogen and antiestrogen in the human breast cancer cell line MCF-7

Regulation of breast tumor proliferation depends in a large part on a variety of hormones and growth factors. In this report we show that estrogen and antiestrogen modulate epidermal growth factor-receptor (EGF-R) level in the human breast cancer MCF-7 cells with opposite mechanisms. Although a short-term treatment (24h to 48h) with estradiol leads to a decrease in EGF-R number, the addition of hormone in cell culture for 5 days increases EGF-R level with a maximal effect observed at 10(-10) M estradiol. In contrast, when cells are treated with the antiestrogen hydroxytamoxifen, a dose-dependent decrease in EGF-R level occurs. We also report that EGF is able to induce estrogen receptors and, to a lesser extent, progesterone receptors when added to MCF-7 cell cultures. These results demonstrate an interaction between both estrogen receptor and EGF receptor growth promoting systems in target cells. The implications of such an interaction in the understanding of human breast cancer hormone responsiveness and, in the development of therapies, are discussed.     

Invasive and angiogenic phenotype of MCF-7 human breast tumor cells expressing human cyclooxygenase-2

To evaluate the direct effect of human cyclooxygenase-2 (hCox-2) on human breast tumor cell proliferation, invasion, and angiogenesis, hCox-2 cDNA was transfected into slow growing, non-metastatic MCF-7 human breast tumor cells that express low levels of Cox-2. Two stable transfectant clones, designated MCF-7/hCox-2 clones 8 and 10, had significantly decreased (P < 0.05) doubling time, with two-fold greater number of cells during exponential growth compared to the MCF-7/vector control. Proliferation of both of the MCF-7/hCox-2 clones was significantly inhibited in a time- and dose-dependent manner by celecoxib. The MCF-7/hCox-2 clones 8 and 10 formed larger and greater numbers of colonies in soft agar than the MCF-7/vector control, with a corresponding increased invasion across an artificial Matrigel basement membrane in response to recombinant human epidermal growth factor (hEGF). The MCF-7/hCox-2 clones 8 and 10 had higher mRNA levels of two splice variants of vascular endothelial growth factor (VEGF), V145 and V165. These results demonstrate that hCox-2 directly increases breast tumor cell proliferation, stimulates invasion across a basement membrane, and induces synthesis of specific heparin binding splice variants of VEGF.