Epidermal Growth Factor Receptor In Cells Research Articles

Quantitative comparison of EGFR expression levels of optically trapped individual cells using a capacitance biosensor

Cellular endocytosis is an essential phenomenon which induces cellular reactions, such as waste removal, nutrient absorption, and drug delivery, in the process of cell growth, division, and proliferation. To observe capacitance responses upon endocytosis on a single-cell scale, this study combined an optical tweezer that can optically place a single cell on a desired location with a capacitance sensor and a cell incubation chamber. Single HeLa cancer cell was captured and moved to a desired location through optical trapping, and the single-cell capacitance change generated during the epidermal growth factor (EGF) molecule endocytosis was measured in real time. It was found that single HeLa cells showed a larger increase in capacitance values compared to that of the single NIH3T3 cells when exposed to varying EGF concentrations. In addition, the capacitance change was in proportion to the cell's EGF receptor (EGFR) level when cells of different levels of EGFR expression were tested. An equation derived from these results was able to estimate the EGFR expression level of a blind-tested cell. The biosensor developed in this research can not only quickly move a single cell to a desired location in a non-invasive manner but also distinguish specific responses between cancer and normal cells by continuous measurement of real-time interactions of a single cell in culture to the external ligands.

Biphasic changes in airway epithelial cell EGF receptor binding and phosphorylation induced by components of hogbarn dust

Purpose of the study: Workers in enclosed hogbarns experience an increased incidence of airway inflammation and obstructive lung disease, and an aqueous hogbarn dust extract (HDE) induces multiple inflammation-related responses in cultured airway epithelial cells. Epidermal growth factor receptor (EGFR) phosphorylation and activation has been identified as one important mediator of inflammatory cytokine release from these cells. The studies here investigated both early and late phase adaptive changes in EGFR binding properties and subcellular localization induced by exposure of cells to HDE. Materials and Methods: Cell surface EGFRs were quantified as binding to intact cells on ice. EGFR phosphorylation, expression, and localization were assessed with anti-EGFR antibodies and either blotting or confocal microscopy. Results: In BEAS-2B and primary human bronchial epithelial cells, HDE induced decreases in cell surface EGFR binding following both 15-min and 18-h exposures. In contrast, H292 cells exhibited only the 15-min decrease, with binding near the control level at 18 hr. Confocal microscopy showed that the 15-min decrease in binding is due to EGFR endocytosis. Although total EGFR immunoreactivity decreased markedly at 18 hr in confocal microscopy with BEAS-2B cells, immunoblots showed no loss of EGFR protein. HDE stimulated EGFR phosphorylation at both 15 min and 18 hr in BEAS-2B cells and primary cells, but only at 15 min in H292 cells, indicating that the different EGFR binding changes among these cell types is likely related to their different time-dependent changes in phosphorylation. Conclusions: These studies extend the evidence for EGFRs as important cellular targets for components of HDE and they reveal novel patterns of EGFR phosphorylation and binding changes that vary among airway epithelial cell types. The results provide both impetus and convenient assays for identifying the EGFR-activating components and pathways that likely contribute to hogbarn dust-induced lung disease in agricultural workers.

Prostaglandin E2-stimulated prostanoid EP4 receptors induce prolonged de novo prostaglandin E2 synthesis through biphasic phosphorylation of extracellular signal-regulated kinases mediated by activation of protein kinase A in HCA-7 human colon cancer cells

Approximately two decades have passed since E-type prostanoid 4 (EP4) receptors were cloned, and the signaling pathways mediated by these receptors have since been implicated in cancer development through the alliance of Gαi-protein/phosphatidylinositol 3-kinase (PI3K)/extracellular signal-regulated kinases (ERKs) activation. Although prostanoid EP4 receptors were initially identified as Gαs-coupled receptors, the specific/distinctive role(s) of prostanoid EP4 receptor-induced cAMP/protein kinase A (PKA) pathways in cancer development have not yet been elucidated in detail. We previously reported using HCA-7 human colon cancer cells that prostaglandin E2 (PGE2)-stimulated prostanoid EP4 receptors induced cyclooxygenase-2 (COX-2) as an initiating event in development of colon cancer. Moreover, this induction of COX-2 was mediated by transactivation of epidermal growth factor (EGF) receptors. However, direct activation of EGF receptors by EGF also induced similar amounts of COX-2 in this cell line. Thus, the emergence of unique role(s) for prostanoid EP4 receptors is expected by clarifying the different signaling mechanisms between PGE2-stimulated prostanoid EP4 receptors and EGF-stimulated EGF receptors to induce COX-2 and produce PGE2. We here demonstrated that prostanoid EP4 receptor activation by PGE2 in HCA-7 cells led to PKA-dependent re-activation of ERKs, which resulted in prolonged de novo synthesis of PGE2. Although EGF-stimulated EGF receptors in cells also induced COX-2 and the de novo synthesis of PGE2, the activation of this pathway was transient and not mediated by PKA. Therefore, the novel mechanism underlying prolonged de novo synthesis of PGE2 has provided an insight into the importance of prostanoid EP4 receptor-mediated Gαs-protein/cAMP/PKA pathway in development of colon cancer.

An improved cell membrane chromatography method for the simultaneous screening of two epidermal growth factor receptor antagonists from radix scutellariae.

A high-expression epidermal growth factor receptor cell membrane chromatography using the silica gel with the average particle size of 3 μm as the stationary phase carrier coupled with high-performance liquid chromatography and mass spectrometry was established for the online screening of epidermal growth factor receptor antagonists from Radix Scutellariae (Huang Qin in Chinese), a traditional Chinese medicine. In this study, the growth factor receptor cell membrane chromatography model using the smaller particle size carrier showed a higher efficiency for simultaneous screening baicalein, another one of the potential epidermal growth factor receptor antagonists from Radix Scutellariae extract besides wogonin, which was found in our previous work. The molecular docking result showed the occupancy site and binding mode of baicalein and wogonin with epidermal growth factor receptor tyrosine kinase were similar to gefitinib. The result of the assay for the in vitro inhibitory activity showed that baicalein and wogonin inhibited the growth of the high-expression epidermal growth factor receptor cell in a dose-dependent manner and even achieved a better inhibition effect than gifitinib in the low-dosage range.

Screening target components from Radix salviae miltiorrhiae using an EGFR/CMC-online-HPLC/MS method

Radix salviae miltiorrhizae is a widely used traditional Chinese medicine, it is the root and rhizome of Salvia miltiorrhiza Bge and belongs to the family Labiatae. Many of its pharmacological effects have been reported in a previous study. In this study, a two-dimension online analytical method of epidermal growth factor receptor cell membrane chromatography combined with liquid chromatography/mass spectrometry was built for recognition, separation and identification of active components from traditional Chinese herb Radix salviae miltiorrhizae. Gefitinib was used as positive control, while atenolol, nicotine, and nimodipine were used as negative controls to investigate the selectivity of the EGFR/CMC column and system suitability of this online analytical method. Cryptotanshinone was screened from Radix salviae miltiorrhizae by this online analytical method. Through in vitro pharmacological trials, cryptotanshinone showed obvious and dose-dependent inhibition activity in the range 0.40 to 50.0 μM. Molecular docking assay showed that the cryptotanshinone binding region was in very good agreement with gefitinib. The EGFR/CMC-online-LC/MS two-dimension analytical method was demonstrated as an effective screening method that rapidly recognizes and enriches active components acting on the epidermal growth factor receptor from Radix salviae miltiorrhizae and separates and identifies them.

Minimally invasive colorectal resection is associated with a rapid and sustained decrease in plasma levels of epidermal growth factor (EGF) in the colon cancer setting

Epidermal growth factor (EGF) stimulates tumor growth directly via tumor cell EGF receptors or indirectly via its proangiogenic effects. This study's purpose was to determine the impact of minimally invasive colorectal resection (MICR) on postoperative (postop) plasma EGF levels in the colorectal cancer (CRC) and benign disease settings and to see if preoperative (PreOp) EGF levels are altered in cancer patients. MICR patients with benign pathology (n = 40) and CRC (n = 48) had blood samples taken PreOp and on postoperative days (POD) 1 and 3. In some patients, late samples were taken between POD7 and POD60; these were bundled into 7-day blocks and considered as single time points. EGF levels were determined by enzyme-linked immunosorbent assay (ELISA) and results were reported as mean ± SD after logarithmic transformation. The Student t test was used (p < 0.008 after Bonferroni correction). The cancer and benign groups were comparable except for age. The mean PreOp CRC plasma EGF level (122.9 ± 75.9 pg/ml) was significantly higher than that of the benign group (85.3 ± 38.5 pg/ml) (p = 0.015). The cancer group's EGF levels were significantly decreased on POD1 and POD3 and for the POD31-55 time point (mean EGF level = 63.1 ± 42.2 (n = 10). The benign group's POD3 and POD7-14 EGF levels were significantly lower than the PreOp level; later levels returned toward baseline. Small late sample size limited analysis. Plasma EGF levels are significantly higher in cancer patients. MICR is associated with a significant decrease in EGF levels early postop in both cancer and benign settings. Unlike the benign group, EGF blood levels in cancer patients remain low during the second postop month. A larger study with more late samples is needed to verify these results. EGF may have value as a tumor marker.

Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor

We have prepared and characterized a new fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for fluorescence studies of EGF–EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra of A-EGF makes this probe well suited to fluorescence resonance energy homo-transfer. Using time-resolved fluorescence to examine the oligomeric state of the EGF receptor, we have observed resonance energy homo-transfer of A-EGF bound to EGF receptors in cells, but not of A-EGF bound to EGF receptors in membrane vesicles. Our results, interpreted in the context of recent crystallographic studies of the ligand-binding domains of EGF receptors, suggest that observed fluorescence resonance energy transfer does not result from transfer within receptor dimers, but rather results from transfer within higher-order oligomers. Furthermore, our results support a structural model for oligomerization of EGF receptors in which dimers are positioned head-to-head with respect to the ligand-binding site, consistent with the head-to-head interactions observed between adjacent receptor dimers by X-ray crystallography.

Transgenic overexpression of amphiregulin induces a mitogenic response selectively in pancreatic duct cells

Background & Aims: The epidermal growth factor (EGF) receptor family and the corresponding ligands are frequently overexpressed in pancreatic cancer. To compare the biological effects of transforming growth factor (TGF)-α and amphiregulin (AR) on growth and differentiation of the exocrine pancreas, we have generated transgenic mice overexpressing AR under control of the elastase promoter. Methods: Two independently generated transgenic mouse lines overexpress 50-, 43-, 28-, 26-, and 16-kilodalton AR forms in the pancreas. Results: Morphologic and immunohistochemical examinations suggest that small intralobular duct and centro-acinar cells proliferate in response to AR in these mice. AR transgenic mice display increased Ras, Erk1/2, cyclin D/CDK4, and cyclin E/CDK2 activity and G1/S progression in pancreatic duct cells. In contrast to TGF-α transgenic mice, AR neither induced tubular complex formation nor elicited a strong fibrogenic response. AR induced a slight induction of ErbB2 on duct cells, whereas TGF-α resulted in overexpression of the EGF receptor in cells within tubular complexes. Furthermore, AR and TGF-α displayed different effects on differentiation of isolated acini in vitro comparable to the situation in vivo. Conclusions: These data suggest that AR induces a mitogenic response selectively in small duct cells through activation of Ras, CDK2, and CDK4, respectively. The closely related EGF receptor ligands, AR and TGF-α, display different biological effects when overexpressed in the exocrine pancreas in vivo.GASTROENTEROLOGY 2002;122:1898-1912

Cag+ Helicobacter pylori Induce Transactivation of the Epidermal Growth Factor Receptor in AGS Gastric Epithelial Cells

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find that H. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag+ strains of H. pylori are able to induce greater EGF receptor activation than cag- strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGF-like growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag+ H. pylori infection.

Her4 mediates ligand-dependent antiproliferative and differentiation responses in human breast cancer cells.

The function of the epidermal growth factor receptor (EGFR) family member HER4 remains unclear because its activating ligand, heregulin, results in either proliferation or differentiation. This variable response may stem from the range of signals generated by HER4 homodimers versus heterodimeric complexes with other EGFR family members. The ratio of homo- and heterodimeric complexes may be influenced both by a cell's EGFR family member expression profile and by the ligand or even ligand isoform used. To define the role of HER4 in mediating antiproliferative and differentiation responses, human breast cancer cell lines were screened for responses to heregulin. Only cells that expressed HER4 exhibited heregulin-dependent antiproliferative responses. In-depth studies of one line, SUM44, demonstrated that the antiproliferative and differentiation responses correlated with HER4 activation and were abolished by stable expression of a kinase-inactive HER4. HB-EGF, a HER4-specific ligand in this EGFR-negative cell line, also induced an antiproliferative response. Moreover, introduction and stable expression of HER4 in HER4-negative SUM102 cells resulted in the acquisition of a heregulin-dependent antiproliferative response, associated with increases in markers of differentiation. The role of HER2 in these heregulin-dependent responses was examined through elimination of cell surface HER2 signaling by stable expression of a single-chain anti-HER2 antibody that sequestered HER2 in the endoplasmic reticulum. In the cell lines with either endogenously (SUM44) or exogenously (SUM102) expressed HER4, elimination of HER2 did not alter HER4-dependent decreases in cell growth. These results suggest that HER4 is both necessary and sufficient to trigger an antiproliferative response in human breast cancer cells.

Shc Mediates Ligand-Induced Internalization of Epidermal Growth Factor Receptors

In order to clarify the physiological relevance of the interaction between Shc and adaptins, components of plasma membrane-coated pit adaptor complex AP2, we investigated the role of Shc in ligand-induced endocytosis of epidermal growth factor (EGF) receptors. In vitro peptide binding assay showed that α-adaptin bound to the wild-type peptide corresponding to amino acids 346–355 of Shc, RDLFDMKPFE, but not to the mutant peptide in which both phenylalanines at 349 and 354 were substituted for alanines (FA). Using adenovirus vectors carrying a herpes simplex virus epitope-tagged 52-kDa wild-type Shc and Shc FA, we examined the interaction between Shc, AP2, and EGF receptors in intact cells. Alpha-adaptin bound to wild-type Shc in an EGF-dependent manner, whereas EGF-dependent association of α-adaptin with Shc FA was markedly reduced. In addition, EGF increased the amount of α-adaptin coprecipitated with EGF receptors in cells expressing wild-type Shc but not Shc FA. These results suggest that EGF stimulates Shc-AP2 complex formation and association of Shc-AP2 complexes with EGF receptors. Internalization assay showed that 125I-EGF internalization was reduced in cells overexpressing Shc FA. Immunofluorescence study showed that punctate staining along the plasma membrane border as well as punctate pattern characteristic of cytoplasmic vesicles near the plasma membrane was enhanced in cells expressing wild-type Shc. These results suggest, therefore, the implication of Shc in ligand-induced endocytosis of EGF receptors in intact cells.

Epidermal growth factor receptor is modulated by redox through multiple mechanisms

The cellular redox state has been shown to play an essential role in cellular signaling systems. Here we investigate the effects of reductants and H2O2 on the signaling of epidermal growth factor (EGF) in cells. H2O2 induced the phosphorylation of the EGF receptor and the formation of a receptor complex comprising Shc, Grb2, Sos, and the EGF receptor. Dimerization or oligomerization of the EGF receptor was not induced by H2O2. Protein tyrosine phosphatase (PTP) assay showed that H2O2 suppressed dephosphorylation of the EGF receptor in cell lysates, suggesting that inactivation of PTP was involved in H2O2-induced activation of the EGF receptor. In contrast, the reductants N-acetyl-L-cysteine [Cys(Ac)] and dithiothreitol markedly suppressed EGF-induced dimerization and activation of the EGF receptor in cells. In accordance with suppression of the EGF receptor, Cys(Ac) suppressed EGF-induced activation of Ras, phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Dithiothreitol completely inhibited EGF binding and kinase activation of the EGF receptor both in vitro and in vivo. In contrast, Cys(Ac) suppressed high-affinity EGF-binding sites on the cells, but had no effect on low-affinity binding sites. Furthermore, Cys(Ac) did not suppress EGF-induced kinase activation or dimerization of the EGF receptor in vitro, indicating that it suppressed the EGF receptor through a redox-sensitive cellular process or processes. Thus, the EGF receptor is regulated by redox through multiple steps including dephosphorylation by PTP, ligand binding, and a Cys(Ac)-sensitive cellular process or processes.

Characterization of epidermal growth factor receptor function in Lysophosphatidic acid signaling in PC12 cells

Lysophosphatidic acid (LPA) is a lipid metabolite that induces the activation of mitogen-activated protein kinase (MAPK) through binding to the G protein-coupled receptor in a number of cell lines and cultures. Recent studies have revealed that LPA is able to rapidly induce the phosphorylation of MAPK through an epidermal growth factor (EGF) receptor-dependent pathway. We investigated the role of the EGF receptor in the signaling pathway initiated by LPA stimulation in nerve growth factor (NGF)-responsive PC12 cells well known to transiently retract their own neurites upon LPA stimulation. LPA-stimulated MAPK signaling was suppressed by the selective EGF receptor inhibitor and in the dominant negative mutant EGF receptor cell line. As in the EGF signaling pathway, the complex of EGF receptor with adapter proteins Shc and Sos was formed in response to LPA stimulation, suggesting there is an intracellular mechanism for transactivation. A neurite retraction assay was also performed to examine the role of the EGF receptor in PC12 cell differentiation, which related to the involvement of LPA-induced neurite retraction. These results suggest that the receptor tyrosine kinase can be activated in a ligand-independent manner through intracellular crosstalk between the signaling pathways.

The Role of Individual SH2 Domains in Mediating Association of Phospholipase C-γ1 with the Activated EGF Receptor

The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma1 (PLC-gamma1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 >> pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 >> pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.

Downregulation of protein kinase C by phorbol ester increases expression of epidermal growth factor receptors in transformed trophoblasts and amplifies human chorionic gonadotropin production

Epidermal growth factor (EGF) and its homologue, transforming growth factor-α (TGF-α), regulate human Chorionic gonadotropin (hCG) synthesis in the human placenta. The current study was designed to investigate the involvement of the protein kinase C pathway in EGF-mediated hCG-β production by JAr choriocarcinoma cells. Downregulation of protein kinase C activity by chronic exposure to the phorbol ester, phorbol 12,13-dibutyrate (PDB), produced a greater increase in hCG-β secretion than did activation of protein kinase C activity by short-term exposure to PDB. Pretreatment with the protein kinase C inhibitors calphostin and chelerythrine also resulted in enhanced basal and EGF-stimulated hCG-β production. Individual concentrations (5 n m EGF and 500 n m PDB) that maximally stimulated hCG production, were additive in combination. The additive effect of PDB on EGF-induced hCG-β secretion was mediated in part by increased JAr cell EGF-receptor concentrations detected by Western blot and Scatchard analyses. The results suggest that EGF and PDB stimulate hCG production in JAr cells by different but interactive mechanisms. It is speculated that downregulation of protein kinase C stimulates basal and EGF-mediated hCG-β production by uninhibiting other signalling pathways that regulate hCG-β secretion in trophoblasts.

Heparin-binding EGF-like growth factor expression and biological action in human pancreatic cancer cells

The epidermal growth factor (EGF) receptor is activated by EGF and other EGF-like growth factors, including heparin-binding epidermal growth factor-like growth factor (HB-EGF). We characterized the biological actions of HB-EGF in PANC-1 and COLO-357 human pancreatic cancer cell lines, and determined whether the presence of HB-EGF in human pancreatic carcinomas correlates with patient survival. HB-EGF enhanced the growth of both cell lines in a dose-dependent manner, with a potency that was generally similar to that of EGF and transforming growth factor-alpha (TGF-alpha). HB-EGF also readily induced tyrosine phosphorylation of the EGF receptor in these cells. Immunohistochemical analysis of 47 pancreatic cancer tissues revealed the presence of HB-EGF immunoreactivity in the cancer cells in 50% of the tumors. However, the presence of HB-EGF was not associated with a statistically significant decrease in the post-operative survival period. Furthermore, coexpression of HB-EGF and the EGF receptor was not associated with shorter patient survival. These findings suggest that HB-EGF activates the EGF receptor in human pancreatic cancer cells, but that it is not involved in enhancing the biological aggressiveness of this malignancy in vivo.

Inhibition of epidermal growth factor receptor aggregation by an antibody directed against the epidermal growth factor receptor extracellular domain.

We have examined the perturbation of epidermal growth factor (EGF) receptor-receptor interactions by a monoclonal antibody (13A9) that binds to the receptor extracellular domain. While 13A9 did not inhibit EGF binding, it inhibited energy transfer between fluorescent-labeled EGF molecules bound to receptors in membranes from human A431 cells by 70-100%. This antibody also inhibited EGF-stimulated receptor dimerization in membranes as assessed by chemical cross-linking and Fab fragments of the antibody strongly inhibited the EGF-stimulated dimerization of solubilized receptors when assessed by velocity sedimentation. However, under conditions where 13A9 inhibited receptor-receptor interactions within the plasma membranes, the antibody had no effect on EGF-stimulated receptor autophosphorylation or tyrosine kinase activity toward an exogenous substrate. Moreover, although the antibody significantly inhibited receptor dimerization in A431 cells, it had no effect on EGF-stimulated changes in cytosolic free [Ca2+] or 125I-EGF uptake in these cells, or on EGF-stimulated DNA synthesis in Swiss 3T3 cells. We conclude that the dimerization of the EGF receptors in a membrane environment is not required for full activation of tyrosine kinase activity and that inhibition of the dimerization of a large fraction of EGF receptors in cells does not necessarily inhibit several EGF-mediated cellular responses.

Regulation of epidermal growth factor receptor expression in normal and transformed keratinocytes

Transformed keratinocytes (SCC-4, SCC-15, SCC-12F2, SVK14) or normal keratinocytes which differ in their differentiation programme were used to study the regulation of EGF-receptor expression. The capacity of the cells to differentiate was modulated by changing the extracellular calcium concentration. We were able to demonstrate that EGF-receptor expression in normal and transformed keratinocytes depends upon the cell type and one or more levels of regulatory control. At the DNA level, EGF-receptor gene amplification occurred in poorly differentiating cells. At the mRNA level, cells showing EGF-receptor gene amplification expressed elevated mRNA and protein levels when cultured under low Ca2+ conditions. Cells not exhibiting EGF-receptor gene amplification showed equal mRNA expression, regardless the Ca2+ concentration in the culture medium. At the protein level, EGF-receptor protein was decreased in cells exhibiting EGF-receptor gene amplification when extracellular Ca2+ was increased (to 1.6 mM) to stimulate differentiation, the decrease in protein being comparable to mRNA expression. Cells not exhibiting EGF-receptor gene amplification showed equal protein expression, regardless of the Ca2+ concentration in the culture medium. Under the same conditions, SV40 transformed keratinocytes showed equal mRNA but elevated protein expression in cells grown under low Ca2+ conditions. At the membrane level, normal keratinocytes and SCC-17F2 cells showed elevated numbers of cell surface exposed EGF-receptors in cells grown under low Ca2+ conditions, but equal mRNA and protein expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Polysomy of chromosome 7 is associated with amplification and overexpression of the EGF‐receptor gene in a human carcinoma cell line derived from a brain metastasis

Overexpression of the EGF-receptor gene is associated with the malignant nature of some tumors. We have recently reported the establishment of a human carcinoma cell line (T-CAR1), derived from a brain metastasis, that had 7 million EGF receptors per cell and was growth inhibited by EGF. The present study was carried out in order to further characterize the EGF-receptor protein in T-CAR1 cells, and to see if the overexpression of the EGF-receptor gene in these cells was associated with abnormalities at the genomic level. We have compared the T-CAR1 cells with the human glioblastoma cell line T-MG1, which has 135,000 EGF-receptors and is growth stimulated by EGF. The MW of the EGF receptors in T-CAR1 cells and T-MG1 cells was estimated to be 170 kDa, equal to the normal EGF-receptor. However, in T-CAR1 cells an additional protein reacted with the monoclonal antibody directed against the internal domain of the EGF receptor. The levels of EGF receptor-related RNAs in T-CAR1 cells and T-MG1 cells reflected the number of EGF receptors in these cell lines. The EGF-receptor gene was amplified ten-fold in T-CAR1 cells, while it was not amplified in T-MG1 cells. No restriction fragment length polymorphism of DNA digested with various restriction enzymes was seen in either of the cell lines. Chromosomal analysis of T-CAR1 cells showed polysomy of chromosome 7 and marker chromosomes derived partly from chromosome 7. Thus, in the T-CAR1 cell line it was an association between polysomy of chromosome 7 and EGF-receptor gene amplification.

Immunohistochemical localization of c‐myc oncogene product and EGF receptor in oral squamous cell carcinoma

Subcellular distribution of c-myc oncogene product and EGF receptor in oral squamous cell carcinomas was examined by using immunohistochemical method. Immunohistochemy for c-myc oncogene showed three staining patterns. Reactive products were demonstrable in the nucleus, in the perinuclear cytoplasm with granular appearance and in the entire cytoplasm. Concomitant stainings of each reactive pattern were observed. Occasionally, the reactive product was strongly deposited on the chromatin under mitotic phase. EGF receptor positive cases were found in only 4 of 28 cases. Though reactive products were usually found in the cytoplasm, the cell surface staining was detected in the only one case. In this case, cells under mitotic phase were strongly stained. The fact that the strong staining both of c-myc and EGF receptor in cells under mitotic phase suggested the close relation between the expression of these genes and the proliferation of carcinoma cells.