Epidermal Growth Factor Binding Studies Research Articles

X-Radiation-Induced Down-Regulation of the EGF Receptor in Primary Cultured Rat Hepatocytes

Exposure to X radiation is associated with a decline in the proliferative activity of the liver, but the molecular mechanism(s) is not well understood. We investigated whether exposure to X radiation is involved in functional changes in the epidermal growth factor (EGF) receptor (EGFR), thereby causing a reduction of EGF-induced DNA synthesis using periportal hepatocytes (PPH) and perivenous hepatocytes (PVH), which differ in their proliferative activity. X radiation dose-dependently decreased DNA synthesis in both subpopulations. The rate of decline in the DNA synthesis was greater in PPH than in PVH, but the zonal difference disappeared after exposure to 10 Gy X radiation. [(125)I]EGF binding studies indicated that high-affinity EGFRs in both subpopulations were down-regulated after X irradiation. Furthermore, EGF-induced EGFR dimerization and phosphorylation at Y1173 in both subpopulations were down-regulated after X irradiation, and the rate of decline was greater in PPH than in PVH. In contrast, phosphorylation at Y845 after EGF treatment was dose-dependently up-regulated after X irradiation in both subpopulations. These results suggest that the X-radiation-related decline in EGF-induced DNA synthesis is caused at least partly by the modification of EGFR function.

Effect of aging on EGF-induced proliferative response in primary cultured periportal and perivenous hepatocytes

Aging relates to declined proliferative capacity of the liver, but the molecular mechanism is not well understood. We examined whether functional changes of epidermal growth factor (EGF) receptor (EGFR) are involved in age-related decline in EGF-induced DNA synthesis using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in the proliferative capacity. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) in 7-, 30-, and 90-week-old rats were isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [methyl-(3)H]thymidine incorporation. EGFR binding affinity to EGF was analyzed by Scatchard analysis using [(125)I]EGF. EGFR dimerization and phosphorylation were determined by Western blot analysis. EGF-induced DNA synthesis was greater in PPH than in PVH from rats of 7 weeks, but the zonal difference disappeared with aging. [(125)I]EGF binding studies indicated that high-affinity EGFR in both subpopulations also disappeared with aging. Furthermore, EGF-induced dimerization in both subpopulations was down-regulated with aging, and the pattern of EGFR phosphorylation was parallel to that of dimerization. These data suggest that age-related decline in EGF-induced DNA synthesis of PPH and PVH is caused by down-regulation of EGFR dimerization through the decrease of high-affinity EGFR.

Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytes

The influence of transglutaminase 2 (TG2) activity on the proliferative effect of epidermal growth factor (EGF) and on EGF receptor affinity in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) has been investigated using a primary culture system. PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF-induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high-affinity EGF receptor expression was markedly up-regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF-induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high-affinity EGF receptor was down-regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down-regulation of high-affinity EGF receptors.

Reduced survival of rectal cancer patients with increased tumor epidermal growth factor receptor levels.

The epidermal growth factor receptor and its various ligands (epidermal growth factor, transforming growth factor-alpha, amphiregulin, heparin-binding epidermal growth factor, heregulin, and betacellulin) have been implicated in growth and regeneration of intestinal mucosa and might be related to the development and progression of gastrointestinal tumors. Although some studies have investigated levels of epidermal growth factor receptor by radioligand binding studies, none of them have further analyzed these levels in patients with rectal cancer and investigated their prognostic value. We quantitatively determined tumor epidermal growth factor receptor levels in 38 patients with colorectal cancer compared with adjacent normal mucosa by iodine-125-labeled epidermal growth factor binding studies and Scatchard analysis. Patients were followed up for 49.5 +/- 32.2 (range, 2-120) months. Epidermal growth factor receptor capacity was increased in invasive colorectal carcinomas according to T classification (P < 0.001), tumors with lymph node infiltration (P = 0.038), and advanced International Union Against Cancer stage (P < 0.001). Survival of colorectal cancer was reduced in patients with advanced International Union Against Cancer stage (P < 0.001), tumors with positive lymph nodes (P < 0.001), and tumors with elevated epidermal growth factor receptor levels (P = 0.024). In rectal cancer patients, poor prognosis was associated with advanced International Union Against Cancer stage (P = 0.029), tumors with lymph node infiltration (P = 0.040), and increased epidermal growth factor receptor levels (P = 0.002). Multivariate Cox regression analysis indicated that elevated levels of epidermal growth factor receptor were an independent predictor of reduced survival in patients with rectal cancer (P = 0.005). The epidermal growth factor receptor/ligand system appears to be involved in tumor development and tumor progression of colorectal carcinomas, with prognostic implication especially in patients with invasive rectal carcinomas. These patients might take advantage of therapies that specifically block epidermal growth factor receptor-mediated signal transduction.

ONCOSTATIN M SUPPRESSES EGF-MEDIATED PROTEIN TYROSINE PHOSPHORYLATION IN BREAST CANCER CELLS

The effect of oncostatin M (OM) on epidermal growth factor (EGF)-mediated protein tyrosine phosphorylation in an infiltrating ductal breast carcinoma cell line, H3922, was investigated by Western blot analysis. Pretreatment of H3922 cells with OM for 72h suppressed EGF-stimulated protein tyrosine phosphorylation signals by 77%. Interestingly, pretreatment with OM for 6 or 48h had little effect on these signals. EGF-mediated tyrosine phosphorylation of EGF receptor (EGFR) was suppressed by 55% in 72-h OM pretreated H3922 cells. No reduction in EGFR protein expression was detected in these cells. Flow cytometric analysis verified that OM does not suppress EGFR expression. The effect of OM could not be attributed to induction of protein tyrosine phosphatases. An H3922 subclone cell line, designated H3922-8, was found to exhibit no proliferative response to treatment with EGF. However, EGF-mediated protein tyrosine phosphorylation was detected in these cells. Radioligand EGF binding studies comparing H3922 to H3922-8 cells indicated that the clonal cells apparently lack high affinity EGF receptors. The mechanism by which OM suppresses EGF-mediated tyrosine phosphorylation has not been completely characterized. However, the suppressive effect occurs regardless of whether the cells are acutely responsive (H3922) or virtually unresponsive (H3922-8) to EGF stimulation of cell growth.

Fibroblasts derived from tissue explants of dilantin-induced gingival hyperplasia and idiopathic gingival fibromatosis show distinct disparity in proliferative responsiveness to epidermal growth factor.

Human gingival fibroblasts derived from tissue explants of two patients with dilantin-induced gingival hyperplasia (DGH) and one patient with idiopathic gingival fibromatosis (GF) were studied with respect to the effect of epidermal growth factor (EGF) on the proliferative characteristics of these cells. Immunohistochemical staining showed that there were more EGF receptor-positive cells among DGH fibroblasts than among either normal gingival fibroblasts (NG) or GF cells. Furthermore, EGF binding studies showed that, in spite of there being no disparity in binding affinity among all these cells, DGH fibroblasts possessed approximately two-fold more EGF receptors than either NG or GF cells. In addition, the growth-promoting effect of exogenously added EGF was concentration-dependent in DGH fibroblasts but was not in either NG or GF cells. All of the above findings clearly demonstrate that DGH and GF fibroblasts exhibit distinct disparity in proliferative responsiveness to EGF and suggest that different mechanisms may be involved in the pathogenesis of these two forms of gingival hyperplasia. These observations also suggest a possible therapeutic approach for blocking EGF-induced cell proliferation in DGH.

EGF causes hyperproliferation and apoptosis in T51B cells: Involvement of high and low affinity EGFR binding sites

We have recently described an in vitro model for liver homeostasis which involves growing T51B rat liver epithelial cells with 1 nM epidermal growth factor (EGF). There is an initial period of hyperplasia, lasting about 3 days, which is followed by an increase in apoptosis. The cell density returns to around the confluent control level 5 days after EGF addition. The dose response of T51B cells to EGF shows three distinct growth patterns. We have carried out EGF binding studies that suggest that the occupancy of the low affinity binding site of the EGF receptor, is responsible for the hyperproliferation seen when the cells are grown with high doses of EGF. These studies also suggest that the apoptosis could be triggered by down-regulation of the receptor, in a manner analogous to the removal of a trophic hormone in other systems.

Cellular distribution of epidermal growth factor, transforming growth factor‐α, and epidermal growth factor receptor in fascia and peritoneum during healing in the rat: an autoradiographic and immunohistochemical study

The presence and cellular distribution of epidermal growth factor (EGF), TGF-alpha, and EGF-R were determined in the rat fascial and peritoneal tissue during healing of an incisional injury by means of immunohistochemistry and autoradiographic techniques. The immunostaining intensity for EGF in the regenerating wound area was substantially higher during the first 14 days, then decreased to near prewound levels during 14 to 35 days after surgery. Within the wound area, the most intense immunostaining occurred with inflammatory cells, followed by fascial striated muscle and arterioles, whereas fibroblasts in the regenerating area contained very low immunostaining intensity. The immunostaining pattern for TGF-alpha with the use of three separate polyclonal antibodies that were directed against the amino and carboxy termini of TGF-alpha precursor and a fragment of the mature 50-amino-acid form of TGF-alpha was similar to that seen with EGF and persisted until 28 days after injury. However, fibroblasts in the regenerating area immunostained intensely for TGF-alpha but not for EGF. Quantitative autoradiography of iodine 125-labeled EGF binding and immunohistochemical studies of the EGF-R with monoclonal antibodies that were directed against the extracellular binding domain of EGF-R demonstrated the presence of specific EGF-R in regenerating fascial and peritoneal tissue. Net grain density (100 microm(2)), representing specific binding of (125)I-EGF, was calculated for different cell types in the wound. The grain density over fascial striated muscle, migratory fibroblasts and peritoneal fibroblasts increased by two and one half, three, and four times, respectively, at 7 days and decreased to the values in adjacent unwounded tissue by 21 days after injury (p < 0.05). Immunostaining for the EGF-R generated similar patterns, which persisted for 14 days after injury. The grain density and immunostaining for EGF-R over the arterioles in the wound did not change during the course of healing and was similar to that of the uninjured regions. In summary, these observations indicate that the local levels of EGF, TGF-alpha, and EGF-R increase during the early phases of healing in fascial and peritoneal injury, which suggests a role for these growth factors in the normal mechanism of fascial/peritoneal wound healing and fibrous adhesion formation.

Triiodothyronine enhances renal tubule cell replication by stimulating EGF receptor gene expression.

Thyroid hormone is known to accelerate renal function recovery following toxic acute renal failure. Because epidermal growth factor (EGF)-receptor activation is most likely critical in renal replicative repair, these studies were undertaken to assess whether triiodothyronine (T3), the most active form of thyroid hormone, may modulate EGF-induced renal proximal tubule cell proliferation by an effect on the EGF receptor. Rabbit renal proximal tubular cells were grown in primary culture and treated with or without T3 (0.1 or 1.0 nM) for 24 to 48 h. Compared with nontreated controls, T3 exposure led to significant increases in EGF-promoted DNA synthesis, as measured by [3H]thymidine incorporation, in renal tubule cells. Furthermore, T3 treatment resulted in increases in EGF receptor mRNA in proximal tubule cells compared with nontreated cells and was associated with elevated numbers of EGF receptors on the cell surface of proximal tubule cells. EGF binding studies demonstrated that T3 treatment had only modest effects on Kd values of both the high-affinity binding site (0.19 nM) and the low-affinity binding site (3.7 nM) but substantially increased the maximal number of high-affinity sites from 3.8 x 10(3) to 9.4 x 10(3) receptors/cell and the maximal number of low-affinity sites from 132 x 10(3) to 199 x 10(3) receptors/cell. These findings suggest that a T3 effect to increase EGF receptor gene expression with resulting increases in the number of cell surface EGF receptors on renal proximal tubule cells and a potentiated mitogenic response to EGF may be a mechanism for thyroid hormone to enhance renal function recovery following toxic acute renal failure.

Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells.

Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 x 10(4) sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 x 10(6) sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1 x 10(-3) s-1 (1.0-1.3 x 10(6) sites per cell) and a slow-dissociating site, with a k-1 of 3.5 x 10(-5) s-1 (0.6-0.7 x 10(6) sites per cell). The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that approximately 5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 x 10(-3) s-1) and a slow-dissociating component (k-1 = 9.1 x 10(-5) s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4 degrees C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4 degrees C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.

Transforming growth factor beta 1 and cAMP inhibit transcription of epidermal growth factor- and oncogene-induced transin RNA.

Transin mRNA encodes a secreted metalloprotease which is transcriptionally induced in Rat-1 cells by epidermal growth factor (EGF) and a number of oncogenes. A role for transin in tumor progression is suggested by its overexpression in malignant and metastatic tumors compared to their benign counterparts. In an effort to elucidate mechanisms by which elevated transin expression may be inhibited, it has been determined that both transforming growth factor type beta 1 (TGF beta 1) and increased levels of intracellular cyclic 5'-adenosine monophosphate (cAMP) inhibit EGF and oncogene induction of transin mRNA. The inhibition of transin mRNA occurred at the level of transcription as demonstrated by nuclear run-on assays. EGF binding studies in Rat-1 cells showed no significant effect of cAMP or TGF beta 1 on EGF receptor number or affinity. We have also examined the effects of cAMP and TGF beta 1 on oncogene-induced transin using Rat-1 cells transformed by temperature-sensitive mutants of v-src and K-ras oncogenes. Both inhibitors prevented the induction of transin RNA as well as decreased the levels of transin once elevated at the permissive temperature. Despite the similarities in the actions of TGF beta 1 and cAMP on transin gene expression, TGF beta 1 treatment did not significantly elevate intracellular cAMP levels, thus making it unlikely that cAMP is a second messenger system for TGF beta 1 action. These studies suggest that the inhibitory effects of cAMP and TGF beta 1 occur by distinct pathways at the level of gene regulation.

Phenobarbital reduces EGF receptors and the ability of physiological concentrations of calcium to suppress hepatocyte proliferation.

Optimal proliferation of cultured hepatocytes from normal rats occurs in response to epidermal growth factor (EGF) at an extracellular calcium concentration of 0.4 mM, whereas physiological concentrations of calcium have been shown to decrease hepatocyte proliferation. Exposure of hepatocytes in vivo to phenobarbital (PB, 0.1% in the drinking water) reduced significantly the ability of physiological levels of calcium to suppress hepatocyte proliferation. An increased relative ability of hepatocytes to proliferate at physiological calcium concentrations versus that at 0.4 mM calcium was first seen after 3 days of in vivo PB treatment and this effect was maintained during 2 months of exposure. Hepatocytes from short-term PB-exposed animals (i.e. 3-28 days) proliferated at physiological Ca2+ concentrations 2-3 times better than those from control animals. However, after 2 months of continuous PB exposure, cell growth was reduced significantly at all extracellular Ca2+ concentrations investigated. EGF binding studies demonstrated that the biphasic kinetic effect of PB on hepatocyte proliferation was not correlated directly with the steady decline in EGF receptor number or the lack of significant change in receptor binding affinity with duration of PB exposure. These results imply that the effectiveness of PB in tumour promotion may result from its ability to reduce the absolute magnitude of normal hepatocyte proliferation and to alter the growth regulatory effect of extracellular calcium. Further, the results argue that PB effects on hepatocyte proliferation are not mediated simply through regulation of EGF surface receptor number or binding affinity. Additional events of the EGF-induced cascade necessary for hepatocyte proliferation appear to be modified by PB.

Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP.

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.

Epidermal growth factor receptors associated to cytoskeletal elements of epidermoid carcinoma (A431) cells.

The structural interaction of the epidermal growth factor (EGF) receptor and the cytoskeleton of A431 cells has been studied using a monoclonal anti-EGF receptor antibody. This has been done with immunogold labeling using a variety of electron microscopical preparation procedures and EGF binding studies. By providing an image of the membrane-associated cytoskeleton, the dry cleavage method reveals a preferential localization of EGF receptors superimposed upon cytoskeletal filaments. The colocalization of gold particles with cytoskeletal filaments is not affected when pre-labeled cells are extracted with the non-ionic detergent Triton X-100, as visualized by dry cleavage. Using surface replication, this treatment results in visualization of the cytoskeleton. In these latter preparations, it is also observed that EGF receptor-coupled gold particles remain associated with cytoskeletal elements. Moreover, Triton extraction performed before immunogold labeling of EGF receptors demonstrates that isolated cytoskeletons contained binding sites for anti-EGF receptor antibodies. Using stereo micrographs of replica's obtained from these isolated cytoskeletons, it is shown that gold-labeled EGF receptors are exclusively present on the cortical membrane-associated region of the cytoskeleton and not on more intracellular-located filaments. Scatchard analysis of EGF binding to cells fixed with glutaraldehyde and treated with Triton X-100 before and after EGF binding indicates that a high affinity EGF binding site is associated with the Triton X-100 insoluble cytoskeleton.

Two forms of murine epidermal growth factor: rapid separation by using reverse-phase HPLC.

Epidermal growth factor (EGF) has been isolated from acid extracts of C57BL6/J mouse submaxillary glands by using hydrophobic chromatography. High yields of EGF in large amounts (10 mg) can be isolated reliably from the acid extract of the glands in less than 4 hr. The reverse-phase HPLC techniques used to purify the EGF initially yielded what appeared to be a single homogeneous EGF molecule. However, ion pairing reagents (e.g., heptafluorobutyric acid) altered the chromatographic properties, revealing two distinct species: EGF-alpha and EGF-beta. The apparent molecular weights, isoelectric points, and antigenic properties of EGF-alpha and EGF-beta were identical, and both forms stimulated a mitogenic response in 3T3 cells. Analysis of different preparations of purified EGF (commercial and experimental) indicated the presence of EGF-alpha and EGF-beta in constant proportion. Previous EGF binding studies must have used mixtures of 125I-labeled EGF-alpha and 125I-labeled EGF-beta. The two molecules appear to compete for an identical receptor on the cell surface.