Long-term Epidermal Growth Factor Research Articles

Plasticity of Mammary Cell Boundaries Governed by EGF and Actin Remodeling

Defined signals that dictate the architecture of cellular boundaries in confluent cultures are poorly characterized. Here, we report dramatic remodeling, invoked by long-term epidermal growth factor (EGF) withdrawal from mammary-derived MCF10A cells. Such intervention generates an interdigitated, desmosome-rich monolayer, wherein cells project actin-containing protrusions deep into neighboring cells. These changes protect cellular sheets from mechanical disruption and dramatically restrict the freedom of cells to roam within the monolayer. Ectopic expression of activated Rac counteracts interdigitation and induces membrane ruffling, but cells remain confined by their interdigitated neighbors. Interdigitations are rapidly dissolved by acute EGF application in a process that is sensitive to actin depolymerization and myosin II inhibition. These assays for formation and dissolution of interdigitations provide a platform for the dissection of novel signaling pathways that are highly specific to EGF receptor (EGFR) activation.

Epidermal Growth Factor Treatment Switches δ-Opioid Receptor-Stimulated Extracellular Signal-Regulated Kinases 1 and 2 Signaling from an Epidermal Growth Factor to an Insulin-Like Growth Factor-1 Receptor-Dependent Mechanism

δ-Opioid receptor (DOR)-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) is mediated by the transactivation of epidermal growth factor (EGF) receptors. Here we demonstrate that in stably DOR-expressing human embryonic kidney (HEK) 293 (HEK/DOR) cells, down-regulation of EGF receptors by long-term EGF (0.1 μg for 18 h) treatment, but not by small interfering RNA, results in functional desensitization of EGF (10 ng/ml)-stimulated ERK1/2 signaling. In EGF receptor-desensitized (HEK/DOR(-EGFR)) cells, however, [d-Ala²,d-Leu⁵]enkephalin (1 μM) and etorphine (0.1 μM) retained their ability to stimulate ERK1/2 activation. The newly acquired signal transduction mechanism is insensitive to the EGF receptor blockers 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785), does not involve DOR internalization and activation of the focal adhesion kinase pp125FAK, but requires matrix metalloproteinase-dependent release of soluble growth factors. A supernatant transfer assay in which conditioned growth media of opioid-treated HEK/DOR and HEK/DOR(-EGFR) "donor" cells are used to stimulate ERK1/2 activity in DOR-lacking HEK293 wild type and HEK293(-EGFR) "acceptor" cells revealed that long-term EGF treatment produces a switch in the receptor tyrosine kinase (RTK) system transactivated by opioids. Using microfluidic electrophoresis, chemical inhibitors, phosphorylation-specific antibodies, and EGF receptor-deficient Chinese hamster ovary-K1 cells, we identified the release of an insulin-like growth factor-1 (IGF-1)-like peptide and activation of IGF-1 receptors in HEK/DOR(-EGFR) cells after DOR activation. A similar switch from a neurotrophic tyrosine kinase receptor type 1 to an IGF-1 receptor-dependent ERK1/2 signaling was observed for chronically nerve growth factor-treated neuroblastoma × glioma (NG108-15) cells. These results indicate that transactivation of the dominant RTK system in a given cellular setting may represent a general feature of opioids to maintain mitogenic signaling.

Epidermal growth factor in clinical practice – a review of its biological actions, clinical indications and safety implications

Chemotaxis, mitogenesis, motogenesis and cytoprotection are common cellular events involved in both tumourigenesis and tissue repair, which appear amplified upon growth factors exposure. Epidermal growth factor (EGF) promotes these events in epithelial and mesenchymal cells through the binding to a specific tyrosine kinase receptor. In experimental oncology settings, EGF does not initiate malignant transformation but exhibits 'tumour promotion'. These observations have raised doubts on the clinical use of EGF despite solid demonstrations of efficacy in experimental conditions and clinical trials. The results of a Pubmed and Bioline investigation on EGF clinical uses and preclinical safety data are presented here. EGF topical administration has been used since 1989 to enhance the healing process of a variety of peripheral tissues wounds (16 clinical reports), as well as its intravenous, oral and rectal administration for gastrointestinal damages (11 clinical reports). EGF therapeutic efficacy and excellent tolerability seem demonstrated. Lack of long-term adverse effects is highlighted in those studies with 6, 12 and 24 months of patients follow-up. Although post-treatment follow-up may fall short for malignant growth, there are no reports on evidences linking EGF clinical use with cancer. A multicentre, nationwide survey in Cuba, 15 years after randomly using silver sulphadiazine with EGF or not in burn victims yielded that cancer incidence was comparable between EGF-treated and control subjects and that such incidence rate does not differ from the age-matched national incidence for those 15-year period. All the animal species subjected to long-term EGF systemic administration exhibit dose-dependent and reversible epithelial organs hyperplasia with no changes in cells phenotypic differentiation. Histotypic pre-malignant markers were not identified. The results emerged from co-carcinogenesis studies and from transgenic mice over-expressing EGF are conflicting and indicate that EGF overexposure, either innate or postnatal, may not be sufficient to transform cells. The ability of EGF to heal injured tissues in life-threatening scenarios or to assist in preventing physical and social disability advocates for its clinical use under a rational medical risk/benefit balance.

Epithelial Mg2+ channel TRPM6: insight into the molecular regulation

Our understanding of the molecular mechanisms of renal magnesium (Mg2+) handling has greatly enhanced over recent years. This review highlights the regulatory factors controlling Mg2+ homeostasis through its effects on the epithelial Mg2+ channel TRPM6 (Transient Receptor Potential Melastatin subtype 6), the gatekeeper of the body's Mg2+ balance. Drug treatment, acid-base status, and several hormones have been shown to regulate TRPM6 expression, while its channel activity is modified by intracellular Mg2+, pH, and ATP. Recently, epidermal growth factor (EGF) and estrogen have been implicated as magnesiotropic hormones. The stimulation of the EGF receptor (EGFR) leads to an intracellular cascade involving Rac1 that promotes trafficking of TRPM6 to the plasma membrane. Furthermore, long-term EGF treatment upregulates the expression of TRPM6. Estrogen has also been shown to stimulate TRPM6 activity upon short-term treatment, next to its long-term regulatory effect on TRPM6 transcription. TRPM6, and its closest homologue TRPM7, are composed of a Mg2+ -permeable channel fused to an alpha-kinase domain. In the intracellular compartment, the receptor for activated C-kinase (RACK1), the repressor for estrogen receptor activity (REA), and ATP were identified as negative modulators of TRPM6 activity through its alpha-kinase domain. Therefore, the a-kinase domain acts as an indirect player involved in Mg2+ homeostasis by its feedback function in the TRPM6-mediated Mg2+ influx.

Long-Term EGF Treatment Partially Prevents Reduction of Renal Blood Flow in Response to Neonatally Induced Partial Unilateral Ureteral Obstruction

Background/Aims: To investigate the effects of long-term epidermal growth factor (EGF) treatment on kidney growth, renal blood flow (RBF), AQP2 expression and urine output in newborn rats with partial unilateral ureteral obstruction (PUUO). Methods: PUUO (n = 29) and sham-operated control (CON) (n = 26) was created in 2-day-old rats. Fifteen PUUO and 14 CON rats were treated with EGF beginning from the second day after operation to 8 weeks of age. Total kidney volume (TKV) and RBF were measured by MRI at weeks 4 and 8, respectively. Urine volume and osmolality, plasma osmolality, and free water clearance were measured. Renal AQP2 expression was determined using semiquantitative immunoblotting. Results: PUUO induced a severe hydronephrosis and RBF reduction in the obstructed kidney which was attenuated in EGF-treated rats. Body weight, urine output and free water clearance increased, osmolality and the osmolar clearance decreased significantly in EGF-treated rats, despite unchanged AQP2 levels. Conclusions: Long-term EGF treatment attenuated the obstruction- induced RBF reduction. Body weight, urine output increased, and urine osmolality decreased in both control and PUUO rats treated with EGF. Solute-free water clearance increased in PUUO rats which seem to be independent of AQP2 expression.

Epidermal growth factor induction of resistance to topoisomerase II toxins in human squamous carcinoma A431 cells

Alteration of the epidermal growth factor (EGF) signaling pathway occurs frequently in human cancer cells and may subsequently affect the cell survival towards anti-cancer agents. To elucidate the effect of long-term EGF treatment on the chemo-sensitivity of human cancer cells, human squamous carcinoma A431 cells (AP) were incubated continuously with 50 ng/ml EGF for 30 weeks and these cells were designated as the AC cells. The long-term EGF treatment did not alter the EGFR level and the EGF-induced protein tyrosine phosphorylation pattern in the AC cells. By MTT assay, the AC cells were shown to be more resistant than the AP cells to doxorubicin, etoposide and amsacrine but not to cisplatin. Among the drug-resistant proteins, topoisomerase IIalpha (topoII) was downregulated in the AC cells while there was no apparent change in the levels of P-glycoprotein, MRP-1 or glutathione- S-transferase-pi as compared to the AP cells. Furthermore, knockdown of topoII by antisense topoII oligonucleotide transfection decreased the sensitivity to doxorubicin, etoposide and amsacrine in the A431 cells. Results from the present study support an idea that long-term treatment with EGF may induce drug resistance in cells through the downregulation of topoII.

Imaging Phosphorylation Dynamics of the Epidermal Growth Factor Receptor

Epidermal growth factor receptor (EGFR) signaling is initiated by ligand binding followed by homodimerization and rapid receptor autophosphorylation. Monitoring EGFR phosphorylation was achieved by measuring translocation and binding of an enhanced yellow fluorescent protein (EYFP)-labeled phosphotyrosine-binding domain (PTB) to enhanced cyan fluorescent protein (ECFP)-tagged EGFR using fluorescence lifetime imaging microscopy or sensitized emission measurements. To simplify dynamic phosphorylation pattern measurements in cells, FLAME, a ratiometric sensor containing both EGFR-ECFP and PTB-EYFP in one molecule, was designed and examined in COS7 cells. Epidermal growth factor (EGF) treatment demonstrated rapid and reversible changes in the EYFP/ECFP fluorescence emission ratios, due to binding of the PTB domain to its consensus binding sites upon phosphorylation at the cell periphery, whereas perinuclear regions failed to respond to EGF but were responsive to tyrosine kinase inhibition. Long-term EGF treatment resulted in accumulation of dephosphorylated receptor in the perinuclear region due to active dephosphorylation occurring at intracellular sites. This indicates that the sensor closely approaches the true dynamics of tyrosine kinase autophosphorylation and dephosphorylation. Phosphatase inhibition by pervanadate resulted in an irreversible response in all cellular compartments. These data show that EGFR is under tonic phosphatase suppression maintaining the receptor in an unphosphorylated (silent) state and is dephosphorylated at endomembranes after ligand-mediated endocytosis.

The effects of epidermal growth factor on gene expression in human fibroblasts.

A better understanding of the molecular effects of epidermal growth factor (EGF) on target cell can help to reveal important aspects of cellular proliferation, transformation, and apoptosis, as well as embryonic and fetal development. In this study, we examined the differences in gene expression of cultured fibroblasts with EGF stimulation for 48 h by using high-density complementary deoxyribonucleic acid (cDNA) arrays. We found that EGF could cause widespread alteration in gene expression. Eight hundred and fifty-five genes, more than 20% of those assayed, showed changed expression, which are involved in various cellular processes, such as energetic metabolism, biosynthesis, the progress of cell cycle, and the signaling pathways of receptor tyrosine kinase (RTKs) and G protein-coupled receptors (GPCRs). The most striking finding is that long-term EGF treatment on cultured fibroblasts resulted in down-regulation of the genes encoding membrane receptors and ion channels and desensitized RTKs and GPCRs to their physiological and nonphysiological stimuli, which seems to be a slow-acting, but permanent, effect of EGF on RTK and GPCR signaling pathways and to play important roles in embryonic and fetal development.

上皮成長因子ＥＧＦが退縮型ラット癌細胞ＥＲ‐１の浸潤，転移能に及ぼす影響 第３報 ＥＧＦ長期間処理による酸化ストレスと遺伝子不安定性の誘導

上皮成長因子ＥＧＦが退縮型ラット癌細胞ＥＲ‐１の浸潤，転移能に及ぼす影響 第３報 ＥＧＦ長期間処理による酸化ストレスと遺伝子不安定性の誘導

Trophic Effects by Epidermal Growth Factor on Duodenal Mucosa and Exocrine Pancreas in Rats

Epidermal growth factor (EGF) is a potent growth factor with possible implications on the regulation of pancreatic secretion and duodenal absorption but also on pancreatic tumor growth. In the present study the growth effect on duodenal mucosa and pancreas by a 14-day continuous infusion of three different doses of EGF (4, 30 and 60 micrograms EGF/kg/24 h) was studied in rats. The EGF content in duodenal mucosa and pancreatic tissue was significantly increased by 30 and 60 micrograms/kg/24 h of EGF while plasma levels were only marginally increased. Neither duodenal mucosal nor pancreatic weights were changed but DNA content in both tissues was increased with the higher EGF doses. Long-term EGF infusion has moderate trophic effect on duodenal mucosa and the pancreas. There is a high tissue uptake of EGF, specially in duodenal mucosa. The hyperplasia seems to be related to tissue levels of EGF but not to plasma levels.

上皮成長因子ＥＧＦが癌細胞の浸潤，転移能に及ぼす影響 第２報 ＥＧＦ長期間処理による悪性形質獲得機序の検討

上皮成長因子ＥＧＦが癌細胞の浸潤，転移能に及ぼす影響 第２報 ＥＧＦ長期間処理による悪性形質獲得機序の検討

Repair of chronic tympanic membrane perforations with long-term epidermal growth factor.

Chronic tympanic membrane (TM) perforation is a common problem worldwide. Recent reports have shown epidermal growth factor (EGF) to stimulate healing in approximately 80% of chronic TM perforations in chinchillas when applied in three doses over 1 week. The objective of this controlled study is to evaluate the efficacy of long-term EGF in the closure of TM perforations. Chronic chinchilla TM perforations were treated with EGF for up to 6 weeks. One hundred percent (17 of 17) of treatment group perforations completely healed. However, two new findings with this long dosing scheme were reperforation on long-term follow-up and three TMs with cholesteatomas. It is likely that reperforation was due to a progressive thinning seen with prolonged EGF application. Long-term EGF use is not recommended for the treatment of TM perforations because of possible wound healing impairment and possible cholesteatoma induction.

上皮成長因子ＥＧＦが癌細胞の浸潤，転移能に及ぼす影響 第１報 ＥＧＦ長期間処理による悪性形質獲得の可能性

上皮成長因子ＥＧＦが癌細胞の浸潤，転移能に及ぼす影響 第１報 ＥＧＦ長期間処理による悪性形質獲得の可能性