EGF Exposure Research Articles

Binding of alpha-ACTN4 to EGF receptor enables its rapid phosphorylation

Alpha-ACTN4, a member of alpha-actinin family is critical for cell motility through its regulated binding of actin filaments. We previously found that EGF exposure of cells triggers the tyrosyl-phosphorylation of ACTN4 in fibroblasts that dramatically downregulates its binding to actin filaments. However, the exact kinase remained uncertain. In the present study, we report that the phosphorylation of ACTN4 occurs within seconds upon EGF treatments and is accomplished via direct interaction of ACTN4 with the EGF receptor. The major binding domain of ACTN4 for EGF receptor is mapped to the N-terminal 32 amino acids. A second domain minimizes the interaction, as truncation of the C-terminal tail enhances ACTN4 binding to EGF receptor. A mimetic phosphorylated ACTN4, Y4/31E, presents low binding to EGF receptor. Overexpression of EGF receptor in melanoma cell lines, also accomplishes the phosphorylation of ACTN4 in the presence of EGF. These findings suggest that the binding of ACTN4 to EGFR enables its direct and rapid phosphorylation resulting in dissociation from EGFR and decreased binding to actin filaments.

EGFR activation attenuates the mechanical threshold for integrin tension and focal adhesion formation.

Mechanical forces, growth factors and the extracellular matrix all play crucial roles in cell adhesion. To understand how epidermal growth factor receptor (EGFR) impacts the mechanics of adhesion, we employed tension gauge tether (TGT) probes displaying the integrin ligand cRGDfK and quantified integrin tension. EGF exposure significantly increased spread area, cell circularity, integrated integrin tension, mechanical rupture density, radial organization and size of focal adhesions in Cos-7 cells on TGT surfaces. These findings suggest that EGFR regulates integrin tension and the spatial organization of focal adhesions. Additionally, we found that the mechanical tension threshold for outside-in integrin activation is tunable by EGFR. Parallel genetic and pharmacologic strategies demonstrated that these phenotypes are driven by ligand-dependent EGFR signaling. Our results establish a novel mechanism whereby EGFR regulates integrin activation and cell adhesion, providing control over cellular responses to the environment.This article has an associated First Person interview with the first author of the paper.

Abstract 2036: In vitro and in vivo effect of carmustine and selenite combination on EGFR signaling in androgen-independent prostate cancer

Abstract Introduction: Despite the use of androgen deprivation therapy, the majority of prostate cancer patients will progress to castration resistant disease within 2-3 years, driven by aberrant androgen receptor activation. We have previously shown that combination of carmustine and selenite effectively induces apoptosis by completely reducing AR and AR-variants in AR-dependent castration resistant prostate cancer cells both in vitro and in vivo. Although studies have demonstrated that AR signaling is a central mechanism of castration resistant prostate cancer progression, the cell growth mediated by AR-independent signaling is also operative. EGFR signaling has been implicated in the survival, invasion, and metastasis of prostate cancer cells in an AR-independent manner. Therefore, in this study, we tested whether the combination of carmustine and selenite could inhibit EGFR signaling, induce apoptosis, and inhibit growth of AR-independent prostate cancer cells in an in vitro cell culture and in vivo xenograft models. Methods: AR negative PC-3 prostate cancer cell line was used to study the effect of carmustine and selenite alone or in combination on EGFR signaling. Cell viability, proliferation, and apoptosis in the presence or absence of EGF (50ng/ml) were determined. Dose and time dependent effect of EGF on the activation of EGFR was studied. The carmustine and/or selenite effect on EGF stimulated phospho-EGFR, and its downstream signaling pathways, such as AKT and MAPK, were studied in comparison with inhibitors of EGFR (AG1478) or PI3 kinase (LY294002) or MAPK (PD98059). The in vivo effect of carmustine and selenite was examined on PC-3 tumor growth in athymic nude mice. Results: Combination of carmustine and selenite treatment in the presence of EGF markedly suppressed cell viability and proliferation of PC-3 cells. EGF exposure increased phospho-EGFR (Tyr845, Tyr992, Tyr1068, and Tyr1045), pAkt (Ser473), and pERK1/2 (Thr204/Tyr202) protein expression levels in PC-3 cells in a dose and time dependent manner. The combination treatment exhibited a strong inhibitory effect on EGF stimulated EGFR, Akt, and ERK1/2 phosphorylation. Combination treatment was able to induce apoptosis even in the presence of EGF. Consistent with in vitro results, combination treatment for 3 weeks reduced PC-3 xenograft tumors in nude mice by 88% without any toxicity. Whereas individual agent treatment showed only partial effect both in vitro and in vivo. Conclusions: Our pre-clinical data demonstrate that the combination treatment of carmustine and selenite substantially inhibits EGFR signaling, induces apoptosis, and reduces tumor growth of AR-independent prostate cancer cells in vitro and in vivo. Our novel findings suggest that the combination of carmustine and selenite is an effective therapeutic agent for successful treatment, survival, and improved quality of life for patients with prostate cancer. Citation Format: Vijayalakshmi Thamilselvan, Mani Menon, Sivagnanam Thamilselvan. In vitro and in vivo effect of carmustine and selenite combination on EGFR signaling in androgen-independent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2036. doi:10.1158/1538-7445.AM2017-2036

Combination of Carmustine and Selenite Inhibits EGFR Mediated Growth Signaling in Androgen-Independent Prostate Cancer Cells.

Although aberrant androgen receptor (AR) signaling is a central mechanism for castration resistant prostate cancer (CRPC) progression, AR-independent growth signaling is also present in CRPC. The current therapeutic options for patients with CRPC are limited and new drugs are desperately needed to eliminate these crucial growth signaling pathways. We have previously shown that combination of carmustine and selenite effectively induces apoptosis and growth inhibition by targeting AR and AR-variants in CRPC cells. High levels of EGFR expression present in the CRPC cells mediates the cell proliferation via AR-independent growth signaling mechanisms. Therefore, in this study, we investigated whether the combination of carmustine and selenite could inhibit EGFR mediated growth signaling and induce apoptosis in androgen independent-AR negative prostate cancer cells. EGF exposure dose and time dependently increased phospho-EGFR (Tyr845, Tyr1068, and Tyr1045), pAkt (Ser473), and pERK1/2 (Thr204/Tyr202) protein expression levels in AIPC cells. Combination of carmustine and selenite treatment markedly suppressed EGF-stimulated proliferation and survival of AIPC cells and effectively induced apoptosis. The ROS generated by the combination of carmustine and selenite exhibited a strong inhibition on EGF stimulated EGFR and its downstream signaling molecules such as Akt, NF-kB, ERK1/2, and Cyclin D1. Individual agent treatment showed only partial effect. Overall, our findings demonstrated that the combination of carmustine and selenite treatment dramatically inhibits EGFR signaling, proliferation, and induces apoptosis in AIPC cells, suggesting a potential candidate for the treatment of CRPC. The results of the study further suggest that the combination of carmustine and selenite treatment can overcome EGFR mediated AR-independent growth response in CRPC during anti-androgen therapy. J. Cell. Biochem. 118: 4331-4340, 2017. © 2017 Wiley Periodicals, Inc.

Abstract 3523: Quantitative measurements of EGFR pathway signaling and modulation in pleural effusion samples from non-small cell lung cancer (NSCLC) patients using single cell network profiling (SCNP).

Abstract Background: SCNP is a multiparametric flow cytometry-based assay that quantitatively and simultaneously measures, at the single cell level, both extracellular surface markers and activation levels of intracellular signaling proteins in response to modulation. SCNP has been successfully applied to hematologic malignancies in both prognostic and predictive analyses. Previous work using bladder washes from bladder cancer patients (pts) established the feasibility of applying SCNP to the functional characterization of epithelial cell signaling and response to modulation and targeted kinase inhibition in non-conventional samples. Objectives: Assess the feasibility of applying SCNP to detect and functionally characterize epithelial cells, including sensitivity to targeted kinase inhibition, in pleural effusion (PE) samples from NSCLC pts. Methods: PE from confirmed NSCLC pts (n=4) were collected at the NSUH and shipped overnight to Nodality for processing on day of receipt. Antibodies to CD45, CytoKeratin (CK), EpCAM, and cPARP were used to differentiate non-apoptotic epithelial cells from leukocytes. DNA aneuploidy was evaluated using the lymphoid population in the patient samples as a diploid G0/G1 reference. Signaling in PI3K and MAPK pathways was quantified through measurement of p-AKT and p-ERK levels at baseline and after in vitro exposure to EGF +/- PI3K inhibitor GDC-0941. The epithelial bladder carcinoma cell line HT-1376 was used as a positive control for epithelial phenotypic staining and EGF-induced signaling. Results: In 3 of 4 PE samples, epithelial cells (DAPI+, CD45-, CK+, and EpCAM+) were identified (range 0.35% to 15.87% of all nucleated cells). Epithelial cells had a DNA index >1.3, indicating aneuploidy and therefore tumor origin. The majority of epithelial cells were cPARP negative (>80%) and suitable for SCNP analysis. In all 3 samples with epithelial cells: 1) EGF modulation induced increased p-AKT and p-ERK; 2) Pre-treatment with GDC-0941 inhibited EGF induced p-AKT but not p-ERK; 3) Constitutive activation of the PI3K pathway in the tumor epithelial cells was suggested by reduction in p-AKT levels compared to basal (no EGF exposure) following GDC-0941 treatment. Conclusion: This study demonstrates the feasibility of applying SCNP to the functional characterization of PE samples from NSCLC pts. Ongoing analyses will expand upon these data, including both additional NSCLC PE samples and the evaluation of compounds targeting alternative signaling pathways relevant to NSCLC (eg: MAPK, EGFR). The feasibility of extending such SCNP analyses to other solid tumor indications, using alternative tissue sources and circulating tumor cells, is under further investigation for both prognostic and response predictive utility. [S.Z. Fields and H. Raftopoulos are senior co-authors.] Citation Format: Matt Westfall, Rachael Hawtin, Todd Covey, Michael Gulrajani, Michelle Atallah, Michelle Cholankeril, Reena Vora, Alessandra Cesano, Scott Z. Fields, Haralambos Raftopoulos. Quantitative measurements of EGFR pathway signaling and modulation in pleural effusion samples from non-small cell lung cancer (NSCLC) patients using single cell network profiling (SCNP). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3523. doi:10.1158/1538-7445.AM2013-3523

Abstract 3067: Regulation of PAX2 expression/activity by hypoxia and EGF in breast cancer cells

Abstract Breast cancer is the most common cancer in women and is associated with the highest mortality rate after lung cancer. The leading cause of deaths associated with this cancer is metastasis, but cellular and molecular mechanisms controlling the metastatic ability of breast cancer cells are only partially elucidated. There is direct and indirect evidence suggesting that PAX2, a transcription factor preferentially activated in breast cancer cells of the luminal subtype, could reduce the invasive and metastatic ability of these cells, by downregulating the expression of ERBB2. It is thus primordial to understand the mechanisms regulating the expression and activity of PAX2 in breast cancer cells. Specifically, because hypoxic conditions prevailing in the center of solid primary tumours are known to regulate the expression of many genes controlling invasion and metastasis, we are investigating the impact of hypoxia-mimicking agent cobalt chloride (CoCl2) on PAX2 expression in breast cancer cells. Similarly, because EGF growth factor regulates the expression and/or activity of many genes controlling invasion and metastasis, we are determining the impact of EGF exposure on PAX2 activation and/or gene expression in ER+ MCF-7 cell line. The identification of factors and mechanisms regulating the expression and activity of PAX2 in breast cancer cells will help understand the biology of this emerging factor in breast cancer cells and may provide multiple strategies to control invasion and metastasis of these cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3067. doi:1538-7445.AM2012-3067

Abstract 1035: WAS/WASL interacting protein family, member 2 (WIPF2) expression in breast carcinomas

Abstract ERBB2 over-expression in breast carcinomas is associated with poor prognosis and early metastatic dissemination. The gene WIPF2 is located on the position q21.2 of the chromosome 17 between ERBB2 and TOP2A (17q21-q22) genes and encodes a WASP interacting protein (WIRE protein). It has been reported that this protein has a role in the WASP-mediated organization of the actin cytoskeleton. This protein is a potential link between the activated platelet-derived growth factor receptor and the actin polymerization machinery and its expression has never been evaluated in cancer tissues. The objectives of this investigation were the characterization of the levels of expression of WIPF2 gene in breast carcinomas and its correlation with EGFR 1 stimuli. Material and Methods: We studied the expression of WIRE protein in 87 ductal invasive carcinomas and WIPF2 gene in three breast cancer cell lines (Hb4a-C5.2, MCF7 and SKBR3). Breast cancer samples were mounted in a TMA block and the immunohistochemistry was performed to evaluate the expression of WIRE protein, estrogen receptor, progesterone receptor, HER-2 and Ki-67. The HER2, WIRE and KI-67 expression was further studied by using ImageScope software after digital image capture (Aperio). Tumors were classified according ER, PgR, HER2 and Ki-67 expression in luminal A or B, HER2 positive and basal-like subtypes. The WIPF2 and ERBB2 genes expression (real time RT-PCR) and WIRE protein (immunofluorescence) was analyzed in all cell lines before and after EGF treatment (40ng/µl for 12 and 72 hours). Results: We observed that WIRE protein is ubiquitously expressed in breast carcinomas. According to digital analysis, HER2 positive tumors have a higher expression of WIRE protein (p = 0.02). SKBR3 cell line (HER2 over expressed) has a significant 11 fold increase in WIPF2 gene expression comparing to MCF7. After EGF (EGFR 1 ligand) treatment, we observed a significant decrease in WIPF2 gene expression in SKBR3. Additionally, we observed a subcellular modification of WIRE protein, specially in SKBR3 cell line. The protein is usually dispersed into the cytoplasm. After EGF treatment, there is a reduction in its expression and the protein concentrate in endoplasmic reticulum (peri-nuclear). The decrease in WIPF2 gene expression and the subcellular protein modification is time dependent. They are more expressive after 72 hours of EGF exposure. Conclusion: WIRE protein is over-expressed in HER2 ductal breast carcinomas. EGF stimuli (EGFR1) changes its sub-cellular localization and reduces the WIPF2 gene expression in SKBR3 (HER2 positive) cell line. It is possible to infer that the expression of WIPF2 could be associated with ERFR signaling and the actin cytoskeleton motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1035. doi:10.1158/1538-7445.AM2011-1035

E2F3a Is Critically Involved in Epidermal Growth Factor Receptor–Directed Proliferation in Ovarian Cancer

We describe for the first time a new integral molecular pathway, linking transcription factor E2F3a to epidermal growth factor receptor (EGFR) activation in ovarian cancer cells. Investigations on the role of E2F family members in EGFR-mediated mitogenic signaling revealed that E2F3a was selectively upregulated following EGFR activation, whereas all other E2F family members remained unaffected. In contrast, EGF treatment of healthy ovarian surface epithelial and mesothelial cells yielded a selective upregulation of proliferation-promoting E2F1 and E2F2 without influencing E2F3a expression. In ovarian cancer cell lines, the extent of EGF-induced proliferative stimulus was closely related to the magnitude of E2F3a increase, and proliferation inhibition by E2F3a knockdown was not overcome by EGF exposure. Furthermore, the EGFR-E2F3a axis was found to be signal transducer and activator of transcription 1/3 dependent and the ratio of IFN-regulatory factor (IRF)-1 to IRF-2 was shown to be determinative for E2F3a control. In a pilot study on 32 primary ovarian cancer specimens, a highly significant correlation between activated EGFR and E2F3a expression was disclosed. This new integral pathway in the EGFR-driven mitogenic cell response, which through its key player E2F3a was found to be essential in triggering proliferation in ovarian cancer cells, provides new insights into EGFR signaling and could represent the basis for appealing new therapeutic approaches in ovarian cancer.

Anti-proliferative effects of γ-tocotrienol on mammary tumour cells are associated with suppression of cell cycle progression

Previous studies have shown that gamma-tocotrienol induces potent anti-proliferative effects on +SA mammary tumour cells in culture; here, investigations have been conducted to determine its effects on intracellular signalling proteins involved in regulating cell cycle progression. +SA cells were maintained in mitogen-free defined media containing 0 or 4 micromgamma-tocotrienol, for 48 h to synchronize cell cycle in G(0) phase, and then they were exposed to 100 ng/ml EGF to initiate cell cycle progression. Whole cell lysates were collected at various time points from each treatment group and were prepared for Western blot analysis. Treatment with 4 micromgamma-tocotrienol significantly inhibited +SA cell proliferation over a 4-day culture period. Moreover, this treatment resulted in a relatively large reduction in cyclin D1, cyclin dependent kinase (CDK)4, CDK2 and CDK6 levels, between 4 and 24 h after EGF exposure. Tocotrienol treatment also resulted in a relatively large increase in CDK inhibitor (CKI) p27, prior to and after EGF exposure, but had little effect on levels of CKIs, p21 and p15. Tocotrienol treatment also induced a large relative reduction in retinoblastoma (Rb) protein phosphorylation at ser780 and ser807/811. These findings strongly suggest that anti-proliferative effects of gamma-tocotrienol are associated with reduction in cell cycle progression from G(1) to S, as evidenced by increased p27 levels, and a corresponding decrease in cyclin D1, CDK2, CDK4, CDK6 and phosphorylated Rb levels.

Presenilin 1 Regulates Epidermal Growth Factor Receptor Turnover and Signaling in the Endosomal-Lysosomal Pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.

P42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M(2) and M(3) receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.

Conjugation to Nedd8 Instigates Ubiquitylation and Down-regulation of Activated Receptor Tyrosine Kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.

Modulation of integrin-linked kinase (ILK) expression in human oesophageal squamous cell carcinoma cell lines by the EGF and TGFbeta1 growth factors.

BackgroundIntegrin-linked kinase (ILK) is a ubiquitously expressed protein kinase that has emerged as one of the points of convergence between integrin- and growth factor-signalling pathways.ResultsIn this study we identify the ILK isoform expressed in five human oesophageal squamous cell carcinoma cell lines of South African origin as ILK1, and demonstrate its cellular distribution. ILK expression, although similar in the majority of the cell lines, did show variation. Furthermore, the ILK expressed was shown to be catalytically functional. The effect of growth factors on ILK expression was examined. An increase in ILK expression, following EGF and TGFβ1 exposure, was a trend across all the five oesophageal carcinoma cell lines tested.ConclusionThese results suggest that growth factor modulation of ILK expression relies on the internalisation/recycling of growth factor receptors and stimulation of the PI3K pathway, which may have implications with regards to cell adhesion and tumourigenesis.

Effects of transdifferentiation and EGF on claudin isoform expression in alveolar epithelial cells.

Rat alveolar epithelial type II cells grown on polycarbonate filters form high-resistance monolayers and concurrently acquire many phenotypic properties of type I cells. Treatment with EGF has previously been shown to increase transepithelial resistance across alveolar epithelial cell (AEC) monolayers. We investigated changes in claudin expression in primary cultured AEC during transdifferentiation to the type I cell-like phenotype (days 0, 1, and 8), and on day 5 in culture +/- EGF (10 ng/ml) from day 0 or day 4. Claudins 4 and 7 were increased, whereas claudins 3 and 5 were decreased, on later compared with earlier days in culture. Exposure to EGF led to increases in claudins 4 and 7 and decreases in claudins 3 and 5. Claudin 1 was only faintly detectable in freshly isolated type II cells and remained unchanged over time in culture and after exposure to EGF. These results suggest that increases in transepithelial resistance accompanying AEC transdifferentiation and/or EGF exposure are mediated, at least in part, by changes in the pattern of expression of specific claudin isoforms.

Selective increase of alpha2-integrin sub-unit expression on human carcinoma cells upon EGF-receptor activation.

The effects of chronic EGF exposure on expression of the alpha2beta1 collagen and alpha5beta1 fibronectin receptor in a pair of human carcinoma cell lines (A431 and A549) with differential responses to EGF in a short-term ECM-cell adhesion assay were investigated. Treatment with EGF at 10 ng/ml for 24 hr increased on both cell lines the expression of the alpha2- but not the beta1- or alpha5-integrin sub-units, and concomitantly cellular adhesion was increased on collagen IV but not on fibronectin. Increased collagen adhesion of A549 cells could be blocked by alpha2- and beta1-integrin-sub-unit antibodies down to control levels, while it was blocked by alpha2-integrin-sub-unit antibody only by 60% and completely by the beta1-integrin-sub-unit antibody on A431 cells. EGF induced disparate shifts in cell morphologies (dome-like structures, A431, vs. spindle-like fibroblastoid, A549) with concomitant opposite changes in the expression/localization of E-cadherin in cell-cell contacts. This could be taken as an indication for cell-type-specific differential changes in the ratio of cell-ECM vs. cell-cell contacts. The EGF-induced up-regulation of the alpha2beta1 integrin was instrumental in increasing collagen adhesion of A549 but only partly in the case of A431 cells, in which cells the alpha2beta1 integrin may have additional functions besides serving as cell-ECM receptor.

Selective increase of α2‐integrin sub‐unit expression on human carcinoma cells upon EGF‐receptor activation

The effects of chronic EGF exposure on expression of the α2β1 collagen and α5β1 fibronectin receptor in a pair of human carcinoma cell lines (A431 and A549) with differential responses to EGF in a short-term ECM-cell adhesion assay were investigated. Treatment with EGF at 10 ng/ml for 24 hr increased on both cell lines the expression of the α2- but not the β1- or α5-integrin sub-units, and concomitantly cellular adhesion was increased on collagen IV but not on fibronectin. Increased collagen adhesion of A549 cells could be blocked by α2- and β1-integrin-sub-unit antibodies down to control levels, while it was blocked by α2-integrin-sub-unit antibody only by 60% and completely by the β1-integrin-sub-unit antibody on A431 cells. EGF induced disparate shifts in cell morphologies (dome-like structures, A431, vs. spindle-like fibroblastoid, A549) with concomitant opposite changes in the expression/localization of E-cadherin in cell-cell contacts. This could be taken as an indication for cell-type-specific differential changes in the ratio of cell-ECM vs. cell-cell contacts. The EGF-induced up-regulation of the α2β1 integrin was instrumental in increasing collagen adhesion of A549 but only partly in the case of A431 cells, in which cells the α2β1 integrin may have additional functions besides serving as cell-ECM receptor. Int. J. Cancer 80:546–552, 1999. © 1999 Wiley-Liss, Inc.

EGF Promotes Development of a Differentiated Trophoblast Phenotype Having c-myc and junB Proto-oncogene Activation

Human placental cytotrophoblast cells differentiate by a process of fusion into a syncytium. This process is stimulated by EGF but also occurs spontaneously at a slower rate in cultured cytotrophoblast cells. To determine nuclear proto-oncogene changes mediating these events, c-myc, c-fos, c-jun and junB were measured in spontaneously differentiating cells and in cells exposed to EGF. c-myc showed a transient rise in expression at 4–8h with augmented expression by EGF, occurring even in the absence of serum or attachment. c-myc and c-jun declined during culture, but c-fos and particularly junB showed increased expression by day 3 with marked responses to EGF stimulation. Syncytia induced to form by EGF exposure for 48h demonstrated marked junB expression after rechallenge with 40min EGF exposure, but negligible responses of c-fos and c-jun. c-myc showed increased expression after 6h EGF exposure throughout the culture period and in syncytia. The results indicate EGF promotes a syncytial phenotype characterized by c-fos and junB expression during syncytial formation. EGF continues to elicit junB and c-myc responsiveness in more mature syncytium, indicative of continued EGF actions which may include acting as a survival factor, as an hCG secretagogue, and as an inducer of continued development of the syncytium.

Modulation of gap junctional intercellular communication by EGF in human kidney epithelial cells.

Modulation of gap junctional intercellular communication (GJIC) was studied in a multistep model of human renal epithelial carcinogenesis. We report that the majority of primary human kidney epithelial cells (NHKE) grown from fetal kidney explants did not communicate through gap junctions. Communication could, however, be observed within a subpopulation of the cells. Ni(II)-immortalized cells (IHKE) showed GJIC at a level of 10-20 communicating cells, but with heterogeneous regions on the dish, with regard to both communication and distribution of connexin43. The heterogeneity was less pronounced in a ras-transfected tumourigenic cell line (THKE), which also showed communication of approximately 10-20 dye-coupled cells. Communication within the IHKE sub-clone K7 decreased from 55 dye-coupled cells communicating on day 1 after seeding to approximately 13 in cells grown for 4 days. Daily change of growth medium reduced the decrease in GJIC. EGF enhanced communication following a lag period which depended on days in culture. The largest increase in GJIC was observed in 2-day-old cultures, where the number of communicating cells in some experiments increased from approximately 45 to 130 dye-coupled cells 4 h following change to medium with EGF. The induction was concentration dependent and communication was enhanced gradually between 2 and 7 h after exposure to EGF. A 15 min pulse of EGF was sufficient to induce the GJIC increase if the total incubation period was unchanged. Cycloheximide completely blocked the EGF-induced enhancement of communication, while actinomycin D had no effect. EGF exposure resulted in an increase in the cellular level of connexin43 protein in parallel with the enhancement in communication. Together, these results indicate that the EGF-induced enhancement of GJIC in human kidney epithelial cells was mediated through translational control of connexin43 expression.

Cell Division Does Not Increase Transepithelial Permeability of LLC-PK1 Cell Sheets

The transepithelial electrical resistance (TER) across LLC-PK1 cell sheets is unstable for several days postseeding, even when the cells are trypsinized from a previously confluent culture and replated at confluent density. We therefore followed the TER of LLC-PK1 cells plated at confluent density to elucidate characteristics of the TER fluctuations after plating of the cells. Control cultures reached a maximum TER of 1800 Ω · cm2 approximately 24 h after plating. TER then declined sharply, reaching generally stable values (≈175 Ω · cm2) only after 4 days. Cell cycle activity ([3H]thymidine incorporation) peaked approximately 22 h after plating, prior to the peak in TER values, and then followed a decline similar to that of the TER. Treatment of cells with EGF at 24 h after plating caused the TER values reached at 3-4 days postseeding to exceed time matched controls by approximately 100%. This EGF-treated group showed a concomitant increase in [3H]thymidine incorporation and cell density compared to control. Transepithelial fluxes of [14C]D-mannitol across control vs EGF-treated cell sheets were not, however, significantly different at the 4-day time point, indicating that a change in tight junction sieving on the basis of size had not occurred. Diffusion and bi-ionic potential studies indicated that the change in TER in EGF-treated cells was instead due to altered charge selectivity at the tight junction and/or intercellular space. We conclude: (1) TER across LLC-PK1 cell sheets does not stabilize until 4 days after seeding at confluent density and (2) cell division and resultant increased cell density in LLC-PK1 cell sheets can correlate with elevated TER values, due to altered ion permeability of the paracellular pathway. Permeability to both Na and Cl decreased as a result of EGF treatment but the decline in chloride permeability was significantly greater. Not only was there a decrease in the permeabilities of all halide anions after exposure of cell sheets to EGF, but the permeability sequence changed after EGF exposure.

191 EPIDERMAL GROWTH FACTOR (EGF) EXTENDS LIFE SPAN OF NORMAL HUMAN FIBROBLASTS IN CULTURE

Normal human fibroblast cultures derived from newborn foreskin (HF) have a limited in vitro life span of 50 ± 10 cumulative population doublings (CPD). Adding EGF (25 ng/ml) to cultures at different stages of in vitro aging increased the survival of individual strains by as much as 25% (10-15 CPD). In early single passages, EGF produced a nearly 2 fold greater cell density over control cultures. With increasing CPD, HF progressively showed decreased EGF sensitivity as reflected by decreased stimulation of cells to initiate DNA synthesis and decline in saturation density of EGF treated cultures. Cell density was similar in control and EGF cultures during the final 4-6 CPD. HF exposed to EGF from early stages of explant outgrowth showed no greater CPD than those exposed to EGF at later stages. Intermittent or discontinuous EGF exposure from early passages produced maximum CPD. Removal of EGF from cultures after early continuous exposure did not abolish the expanded culture survival. After control HF reached about 80% of expected CPD, exposure to EGF did not significantly extend life span of the cultures. Colony size distribution was determined during early, middle and late passages of strains with/without EGF exposure. In each strain regardless of CPD, EGF significantly increased the size distribution curve of derived colonies. Thus, EGF enhanced the survival of cells in subculture and prolonged their proliferative activity significantly beyond control cultures.