EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation

Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.     

Cell-surface perturbations of the epidermal growth factor and vascular endothelial growth factor receptors by phosphorothioate oligodeoxynucleotides

Antisense oligodeoxynucleotides offer potential as therapeutic agents to inhibit gene expression. Recent evidence indicates that oligodeoxynucleotides designed to target specific nucleic acid sequences can interact nonspecifically with proteins. This report describes the interactive capabilities of phosphorothioate oligodeoxynucleotides of defined sequence and length with two essential protein tyrosine receptors, flk-1 and epidermal growth factor receptor (EGFR), and their effects on receptor signaling in a transfected and tumor cell line, respectively. Phosphorothioate oligodeoxynucleotides bound to the cell surface, as demonstrated by fluorescence-activated cell-sorter analyses (FACS), and perturbed receptor activation in the presence and absence of cognate ligands, EGF (EGFR) and vascular endothelial growth factor (flk-1), in phosphorylation assays. Certain phosphorothioate oligodeoxynucleotides interacted relatively selectively with flk-1 and partially blocked the binding of specific anti-receptor monoclonal antibodies to target sites. They stimulated EGFR phosphorylation in the absence of EGF but antagonized ligand-mediated activation of EGFR and flk-1. In vivo studies showed that a nonspecific phosphorothioate oligodeoxynucleotide suppressed the growth of glioblastoma in a mouse model of tumorigenesis. These results emphasize the capacity of phosphorothioate oligodeoxynucleotides to interact with cells in a sequence-selective nonantisense manner, while associating with cellular membrane proteins in ways that can inhibit cellular metabolic activities.     

Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines

In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte.     

Extracellular matrix heparan sulfate modulates endothelial cell susceptibility to Staphylococcus aureus

We have examined several types of tumor cell lines and shown that they invariably expressed little or no Egr-1, in contrast to their normal counterparts. We have previously shown that the expression of exogenous Egr-1 in human breast and other tumor cells markedly reduces transformed growth and tumorigenicity. We therefore hypothesized that the loss of Egr-1 expression plays a role in transformation. All human and mouse breast cancer cell lines and tumors examined had reduced Egr-1 expression compared with their normal counterparts. Reduced Egr-1 expression was also observed in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, and this level increased to normal levels in tumors that regressed after tamoxifen treatment. We concluded, therefore, that loss of Egr-1 expression may play a role in the deregulation of normal growth in the tumorigenic process and that Egr-1 acts as a tumor suppressor gene.     

Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials

The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation upon activation through the binding of one of its ligands. The receptor is located at the cell surface, where the binding of a ligand activates a tyrosine kinase in the intracellular region of the receptor. This tyrosine kinase phosphorylates a number of intracellular substrates that activates pathways leading to cell growth, DNA synthesis and the expression of oncogenes such as fos and jun. EGFR is thought to be involved the development of cancer, as the EGFR gene is often amplified, and/or mutated in cancer cells. In this review we will focus on: (I) the structure and function of EGFR, (II) implications of receptor/ligand coexpression and EGFR mutations or overexpression, (III) its effect on cancer cells, (IV) the development of the malignant phenotype and (V) the clinical aspects of therapeutic targeting of EGFR.     

Differential inhibition by hyperammonemia of the electron transport chain enzymes in synaptosomes and non-synaptic mitochondria in ornithine transcarbamylase-deficient spf-mice: restoration by acetyl-L-carnitine

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.     

Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse

Clara cells are primary targets for metabolically activated pulmonary toxicants because they contain an abundance of the cytochrome P450 monooxygenases required for generation of toxic metabolites. The factors that regulate bronchiolar regeneration after Clara cell injury are not known. Previous studies of naphthalene-induced bronchiolar injury and repair in the mouse have shown that epithelial cell proliferation is maximal 1 to 2 days after injury and complete 4 days after injury. Proliferation is followed by epithelial re-differentiation (4 to 14 days). In this study, mice were treated with the environmental pollutant naphthalene to induce massive Clara cell injury. The distribution and abundance of three growth-regulatory peptides (epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor (TGF)-alpha) was determined immunochemically during repair of this acute bronchiolar injury. EGFR and its ligands were detected at low levels in cells throughout the lung including peribronchiolar interstitial cells, blood vessels, and conducting airway epithelium. Immediately after naphthalene injury (1 to 2 days), EGFR, EGF, and TGF-alpha are expressed in increased abundance in squamous epithelial cells of the injury target zone, distal bronchioles. These immunopositive squamous cells are detected in clumps in the distal bronchioles at the time when cell proliferation is maximal. EGFR protein expression is decreased slightly 4 to 7 days after injury and continues to decrease below control levels of abundance 14 to 21 days after injury. This down-regulation of EGFR is not reflected in a corresponding decrease in EGF and TGF-alpha protein expression, indicating that control of cell proliferation is regulated at the receptor level. Co-localization of EGFR and bromodeoxyuridine-positive proliferating cells in the same bronchiole indicates that EGFR is up-regulated within the proliferative microenvironment as well as in specific proliferating cells within the injury target zone. The coincident localization within terminal bronchioles of EGFR, EGF, and TGF-alpha to groups of squamous epithelial cells 2 days after naphthalene injury suggests that these peptides are important in up-regulating cell proliferation after Clara cell injury in the mouse.